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Sample records for selectively isolate phosphopeptides

  1. The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Larsen, Martin R

    2009-01-01

    , or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding from nonphosphorylated peptides. The enriched phosphopeptides bound to the titanium dioxide are subsequently eluted from the micro-column using an alkaline buffer. Titanium dioxide chromatography is extremely......Titanium dioxide has very high affinity for phosphopeptides and it has become an efficient alternative to already existing methods for phosphopeptide enrichment from complex samples. Peptide loading in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid...... tolerant towards most buffers used in biological experiments. It is highly robust and as such it has become one of the methods of choice in large-scale phospho-proteomics. Here we describe the protocol for phosphopeptide enrichment using titanium dioxide chromatography followed by desalting...

  2. Development of Hf(4+)-immobilized polydopamine-coated magnetic graphene for highly selective enrichment of phosphopeptides.

    Science.gov (United States)

    Lin, Haizhu; Deng, Chunhui

    2016-01-01

    In this work, we first designed and synthesized an IMAC material with Hf(4+) immobilized on polydopamine-coated magnetic graphene and applied it to phosphopeptides enrichment successfully. The newly prepared material gathered the advantages of large specific surface area of graphene, good hydrophilicity and biocompatibility of polydopamine and superparamagnetism of Fe3O4. We investigated the performance of Hf(4+)-immobilized polydopamine-coated magnetic graphene (denoted as magG@PDA-Hf(4+)) in phosphopeptides enrichment and the results showed high selectivity and sensitivity of the new material. Finally, we successfully applied magG@PDA-Hf(4+) to phosphopeptides enrichment from non-fat milk digests and human serum, further demonstrating excellent performance of this new material in phosphopeptides enrichment. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Development of immobilized Sn(4+) affinity chromatography material for highly selective enrichment of phosphopeptides.

    Science.gov (United States)

    Lin, Haizhu; Deng, Chunhui

    2016-11-01

    In this work, we first immobilized tin(IV) ion on polydopamine-coated magnetic graphene (magG@PDA) to synthesize Sn(4+) -immobilized magG@PDA (magG@PDA-Sn(4+) ) and successfully applied the material to highly selective enrichment of phosphopeptides. The material gathered the advantages of large surface area of graphene, superparamagnetism of Fe3 O4 , good hydrophilicity and biocompatibility of polydopamine, and strong interaction between Sn(4+) and phosphopeptides. The enrichment performance of magG@PDA-Sn(4+) toward phosphopeptides from digested β-casein at different concentrations, with and without added digested BSA was investigated and compared with magG@PDA-Ti(4+) . The results showed high selectivity and sensitivity of the Sn(4+) -IMAC material toward phosphopeptides, as good as the Ti(4+) -IMAC material. Finally, magG@PDA-Sn(4+) was applied to the analysis of endogenous phosphopeptides from a real sample, human saliva, with both MALDI-TOF MS and nano-LC-ESI-MS/MS. The results indicated that the as-synthesized Sn(4+) -IMAC material not only has good enrichment performance, but also could serve as a supplement to the Ti(4+) -IMAC material and expand the phosphopeptide coverage enriched by the single Ti(4+) -IMAC material, demonstrating the broad application prospects of magG@PDA-Sn(4+) in phosphoproteome research. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Bioactive Peptides Isolated from Casein Phosphopeptides Enhance Calcium and Magnesium Uptake in Caco-2 Cell Monolayers.

    Science.gov (United States)

    Cao, Yong; Miao, Jianyin; Liu, Guo; Luo, Zhen; Xia, Zumeng; Liu, Fei; Yao, Mingfei; Cao, Xiaoqiong; Sun, Shengwei; Lin, Yanyin; Lan, Yaqi; Xiao, Hang

    2017-03-22

    The ability of casein phosphopeptides (CPPs) to bind and transport minerals has been previously studied. However, the single bioactive peptides responsible for the effects of CPPs have not been identified. This study was to purify calcium-binding peptides from CPPs and to determine their effects on calcium and magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1 to P5 were isolated and the amino acid sequences were determined using LC-MS/MS. Compared with the CPP-free control, all five monomeric peptides exhibited significant enhancing effects on the uptake of calcium and magnesium (P magnesium were presented simultaneously with P5, magnesium was taken up with priority over calcium in the Caco-2 cell monolayers. For example, at 180 min, the amount of transferred magnesium and calcium was 78.4 ± 0.95 μg/well and 2.56 ± 0.64 μg/well, respectively, showing a more than 30-fold difference in the amount of transport caused by P5. These results provide novel insight into the mineral transport activity of phosphopeptides obtained from casein.

  5. Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides.

    Science.gov (United States)

    Zhang, Liyuan; Liang, Zhen; Zhang, Lihua; Zhang, Yukui; Shao, Shujuan

    2015-11-05

    Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga(3+) chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga(3+)-ATP-MNPs). The high specificity of Ga(3+)-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga(3+)-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga(3+)-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga(3+)-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga(3+). Moreover, the unique magnetic core of Ga(3+)-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Facile Preparation of Core-Shell Magnetic Metal-Organic Framework Nanoparticles for the Selective Capture of Phosphopeptides.

    Science.gov (United States)

    Chen, Yajing; Xiong, Zhichao; Peng, Li; Gan, Yangyang; Zhao, Yiman; Shen, Jie; Qian, Junhong; Zhang, Lingyi; Zhang, Weibing

    2015-08-05

    In regard to the phosphoproteome, highly specific and efficient capture of heteroideous kinds of phosphopeptides from intricate biological sample attaches great significance to comprehensive and in-depth phosphorylated proteomics research. However, until now, it has been a challenge. In this study, a new-fashioned porous immobilized metal ion affinity chromatography (IMAC) material was designed and fabricated to promote the selectivity and detection limit for phosphopeptides by covering a metal-organic frameworks (MOFs) shell onto Fe3O4 nanoparticles, taking advantage of layer-by-layer method (the synthesized nanoparticle denoted as Fe3O4@MIL-100 (Fe)). The thick layer renders the nanoparticles with perfect hydrophilic character, super large surface area, large immobilization of the Fe(3+) ions and the special porous structure. Specifically, the as-synthesized MOF-decorated magnetic nanoparticles own an ultra large surface area which is up to 168.66 m(2) g(-1) as well as two appropriate pore sizes of 1.93 and 3.91 nm with a narrow grain-size distribution and rapid separation under the magnetic circumstance. The unique features vested the synthesized nanoparticles an excellent ability for phosphopeptides enrichment with high selectivity for β-casein (molar ratio of β-casein/BSA, 1:500), large enrichment capacity (60 mg g(-1)), low detection limit (0.5 fmol), excellent phosphopeptides recovery (above 84.47%), fine size-exclusion of high molecular weight proteins, good reusability, and desirable batch-to-batch repeatability. Furthermore, encouraged by the experimental results, we successfully performed the as-prepared porous IMAC nanoparticle in the specific capture of phosphopeptides from the human serum (both the healthy and unhealthy) and nonfat milk, which proves itself to be a good candidate for the enrichment and detection of the low-abundant phosphopeptides from complicated biological samples.

  7. Facile synthesis of titania nanoparticles coated carbon nanotubes for selective enrichment of phosphopeptides for mass spectrometry analysis.

    Science.gov (United States)

    Yan, Yinghua; Lu, Jin; Deng, Chunhui; Zhang, Xiangmin

    2013-03-30

    In this work, titania nanoparticles coated carbon nanotubes (denoted as CNTs/TiO2 composites) were synthesized through a facile but effective solvothermal reaction using titanium isopropoxide as the titania source, isopropyl alcohol as the solvent and as the basic catalyst in the presence of hydrophilic carbon nanotubes. Characterizations using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicate that the CNTs/TiO2 composites consist of CNT core and a rough outer layer formed by titania nanoparticles (5-10nm). Measurements using wide angle X-ray diffraction (WAXRD), zeta potential and N2 sorption reveal that the titania shell is formed by anatase titania nanoparticles, and the composites have a high specific surface area of about 104 m(2)/g. By using their high surface area and affinity to phosphopeptides, the CNTs/TiO2 composites were applied to selectively enrich phosphopeptides for mass spectrometry analysis. The high selectivity and capacity of the CNTs/TiO2 composites have been demonstrated by effective enrichment of phosphopeptides from digests of phosphoprotein, protein mixtures of β-casein and bovine serum albumin, human serum and rat brain samples. These results foresee a promising application of the novel CNTs/TiO2 composites in the selective enrichment of phosphopeptides. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Tailor-made magnetic Fe3O4@mTiO2 microspheres with a tunable mesoporous anatase shell for highly selective and effective enrichment of phosphopeptides.

    Science.gov (United States)

    Ma, Wan-Fu; Zhang, Ying; Li, Lu-Lu; You, Li-Jun; Zhang, Peng; Zhang, Yu-Ting; Li, Ju-Mei; Yu, Meng; Guo, Jia; Lu, Hao-Jie; Wang, Chang-Chun

    2012-04-24

    Selective enrichment of phosphoproteins or phosphopeptides from complex mixtures is essential for MS-based phosphoproteomics, but still remains a challenge. In this article, we described an unprecedented approach to synthesize magnetic mesoporous Fe(3)O(4)@mTiO(2) microspheres with a well-defined core/shell structure, a pure and highly crystalline TiO(2) layer, high specific surface area (167.1 m(2)/g), large pore volume (0.45 cm(3)/g), appropriate and tunable pore size (8.6-16.4 nm), and high magnetic susceptibility. We investigated the applicability of Fe(3)O(4)@mTiO(2) microspheres in a study of the selective enrichment of phosphopeptides. The experiment results demonstrated that the Fe(3)O(4)@mTiO(2) possessed remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), large enrichment capacity (as high as 225 mg/g, over 10 times as that of the Fe(3)O(4)@TiO(2) microspheres), extreme sensitivity (the detection limit was at the fmol level), excellent speed (the enrichment can be completed in less than 5 min), and high recovery of phosphopeptides (as high as 93%). In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Fe(3)O(4)@mTiO(2) composite microspheres high benefit for mass spectrometric analysis of phosphopeptides.

  9. Mesoporous TiO2 aerogel for selective enrichment of phosphopeptides in rat liver mitochondria.

    Science.gov (United States)

    Zhang, Liyuan; Liang, Zhen; Yang, Kaiguang; Xia, Simin; Wu, Qi; Zhang, Lihua; Zhang, Yukui

    2012-06-04

    The enrichment of low abundance phosphopeptides before MS analysis is a critical step for in-depth phosphoproteome research. In this study, mesoporous titanium dioxide (TiO(2)) aerogel was prepared by precipitation and supercritical drying. The specific surface area up to 490.7 m(2) g(-1) is achieved by TiO(2) aerogel, much higher than those obtained by commercial TiO(2) nanoparticles and by the latest reported mesoporous TiO(2) spheres. Due to the large specific surface area and the mesoporous structure of the aerogel, the binding capacity for phosphopeptides is six times higher than that of conventional TiO(2) microparticles (173 vs 28 μmol g(-1)). Because of the good compatibility of enrichment procedure with MALDI-TOF-MS and the large binding capacity of TiO(2) aerogel, a detection limit as low as 30 amol for analyzing phosphopeptides in β-casein digest was achieved. TiO(2) aerogel was further applied to enrich phosphopeptides from rat liver mitochondria, and 266 unique phosphopeptides with 340 phosphorylation sites, corresponding to 216 phosphoprotein groups, were identified by triplicate nanoRPLC-ESI-MS/MS runs, with false-positive rate less than 1% at the peptide level. These results demonstrate that TiO(2) aerogel is a kind of promising material for sample pretreatment in the large-scale phosphoproteome study. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Designed synthesis of Graphene @titania @mesoporous silica hybrid material as size-exclusive metal oxide affinity chromatography platform for selective enrichment of endogenous phosphopeptides.

    Science.gov (United States)

    Yao, Jizong; Sun, Nianrong; Deng, Chunhui; Zhang, Xiangming

    2016-04-01

    In this work, a novel size-exclusive metal oxide affinity chromatography (SE-MOAC) platform was built for phosphoproteome research. The operation for preparing graphene @titania @mesoporous silica nanohybrids (denoted as G@TiO2@mSiO2) was facile and easy to conduct by grafting titania nanoparticles on polydopamine (PD)-covered graphene, following a layer of mesoporous silica was coated on the outermost layer. The G@TiO2@mSiO2 nanohybrids exhibited high sensitivity with a low detection limit of 5 amol/μL (a total amount of 1 fmol) and high selectivity for phosphopeptides at a mass ratio of phosphopeptides to non-phosphopeptides (1:1000). The size-exclusive capability of the nanohybrids were also demonstrated by enriching the phosphopeptides from the mixture of Bovine Serum Albumin (BSA), α-casein, and β-casein digests with a high mass ratio (β-casein digests: α-casein: BSA, 1:500:500), which was attributed to the large surface area and ordered mesoporous channels. In addition, the G@TiO2@mSiO2 nanohybrids were employed to capture the endogenous phosphopeptides from human serum successfully. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Hydrophilic Nb{sup 5+}-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xueni; Liu, Xiaodan; Feng, Jianan [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China); Li, Yan, E-mail: yanli@fudan.edu.cn [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China); Deng, Chunhui [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Duan, Gengli [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China)

    2015-06-23

    Highlights: • A new IMAC material (Fe{sub 3}O{sub 4}@PD-Nb{sup 5+}) was synthesized. • The strong magnetic behaviors of the microspheres ensure fast and easy separation. • The enrichment ability was tested by human serum and nonfat milk. • The results were compared with other IMAC materials including the commercial kits. • All results proved the good enrichment ability, especially for multiphosphopeptides. - Abstract: Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe{sub 3}O{sub 4}@polydopamine-Nb{sup 5+} (denoted as Fe{sub 3}O{sub 4}@PD-Nb{sup 5+}) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe{sub 3}O{sub 4}@PD-Ti{sup 4+} microspheres, the Fe{sub 3}O{sub 4}@PD-Nb{sup 5+} microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.

  12. Magnetic MSP@ZrO₂ microspheres with yolk-shell structure: designed synthesis and application in highly selective enrichment of phosphopeptides.

    Science.gov (United States)

    Ma, Wan-Fu; Zhang, Cheng; Zhang, Yu-Ting; Yu, Meng; Guo, Jia; Zhang, Ying; Lu, Hao-Jie; Wang, Chang-Chun

    2014-06-10

    Magnetic yolk-shell MSP@ZrO2 microspheres consisting of a movable magnetic supraparticle (MSP) core and a crystalline ZrO2 shell were synthesized via a two-step controlled "sol-gel" approach for the first time. First, a large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the cross-linked polymethylacrylic acid matrix via a strong hydrogen-bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form a yolk-shell structure, which has better dispersibility and higher adsorbing efficiency of phosphopeptides than its solid counterpart. The formation mechanism of such yolk-shell microspheres could be reasonably proved by the results of TEM, TGA, VSM, XRD, and FT-IR characterization. By taking advantage of the unique properties, the yolk-shell MSP@ZrO2 exhibited high specificity and great capability in selective enrichment of phosphopeptides, and a total of 33 unique phosphopeptides mapped to 33 different phosphoproteins had been identified from 1 mL of human saliva. This result clearly demonstrated that the yolk-shell MSP@ZrO2 has great performance in purifying and identifying the low-abundant phosphopeptides from real complex biological samples. Moreover, the synthetic method can be used to produce hybrid yolk-shell MSP@ZrO2-TiO2.

  13. Characterisation and evaluation of metal-loaded iminodiacetic acid-silica of different porosity for the selective enrichment of phosphopeptides.

    Science.gov (United States)

    Trojer, L; Stecher, G; Feuerstein, I; Lubbad, S; Bonn, G K

    2005-06-24

    Silica particles of different porosity were functionalised with iminodiacetic acid (IDA) and loaded with Fe(III) to yield immobilised metal affinity chromatography stationary phases (Fe(III)-IDA-silica) for phosphopeptide enrichment. The elution step of bound phosphopeptides was optimised with a 32P radioactive labelled peptide by a comprehensive study. Several elution systems, including phosphate buffers of different pH and concentration and ethylenediaminetetraacetic acid solutions were employed. Furthermore the effect of support porosity on elution behaviour was investigated. Under best conditions recoveries higher than 90% were achieved. A solid-phase extraction (SPE) protocol was developed for fractionation of phosphorylated and non-phosphorylated peptides and desalting of the fractions which is essential for subsequent mass spectrometric analysis by the combination of Fe(III)-IDA-silica and C18-silica particles. The pH of the loading buffer was found to be a critical parameter for the efficiency of the SPE protocol. As tryptic digests of alpha-lactalbumin, lysozyme and ribonuclease A mixed with three synthetic phosphopeptides were fractionated, pH 2.5 provided minimal proportion of unspecific bound peptides when comparing the fractions after mu-LC-electrospray ionization MS separation. The effect of a sample derivatisation reaction (methylation) on the efficiency of phosphopeptide enrichment was further investigated. Blocking carboxylate groups by methyl ester formation totally prevented unspecific interaction with the immobilised Fe(III) ions, but generated partially methylated phosphopeptides that increased the complexity of the phosphorylated fraction.

  14. Phosphopeptide enrichment by IEF.

    Science.gov (United States)

    Maccarrone, Giuseppina; Kolb, Nikolaus; Teplytska, Larysa; Birg, Isabel; Zollinger, Richard; Holsboer, Florian; Turck, Christoph W

    2006-11-01

    In our efforts to improve the identification of phosphopeptides by MS we have used peptide IEF on IPG strips. Phosphopeptides derived from trypsin digests of single proteins as well as complex cellular protein mixtures can be enriched by IEF and recovered in excellent yields at the acidic end of an IPG strip. IPG peptide fractionation in combination with MS/MS analysis has allowed us to identify phosphopeptides from tryptic digests of a cellular protein extract.

  15. Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

    Science.gov (United States)

    Hsu, Chuan-Chih; Xue, Liang; Arrington, Justine V.; Wang, Pengcheng; Paez Paez, Juan Sebastian; Zhou, Yuan; Zhu, Jian-Kang; Tao, W. Andy

    2017-06-01

    Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. [Figure not available: see fulltext.

  16. Monodisperse REPO4 (RE = Yb, Gd, Y) hollow microspheres covered with nanothorns as affinity probes for selectively capturing and labeling phosphopeptides.

    Science.gov (United States)

    Cheng, Gong; Zhang, Ji-Lin; Liu, Yan-Lin; Sun, De-Hui; Ni, Jia-Zuan

    2012-02-13

    Rare-earth phosphate microspheres with unique structures were developed as affinity probes for the selective capture and tagging of phosphopeptides. Prickly REPO(4) (RE = Yb, Gd, Y) monodisperse microspheres, that have hollow structures, low densities, high specific surface areas, and large adsorptive capacities were prepared by an ion-exchange method. The elemental compositions and crystal structures of these affinity probes were confirmed by energy-dispersive spectroscopy (EDS), powder X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy. The morphologies of these compounds were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and nitrogen-adsorption isotherms. The potential ability of these microspheres for selectively capturing and labeling target biological molecules was evaluated by using protein-digestion analysis and a real sample as well as by comparison with the widely used TiO(2) affinity microspheres. These results show that these porous rare-earth phosphate microspheres are highly promising probes for the rapid purification and recognition of phosphopeptides. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Ti(IV) carrying polydopamine-coated, monodisperse-porous SiO2 microspheres with stable magnetic properties for highly selective enrichment of phosphopeptides.

    Science.gov (United States)

    Salimi, Kouroush; Usta, Duygu Deniz; Çelikbıçak, Ömür; Pinar, Asli; Salih, Bekir; Tuncel, Ali

    2017-05-01

    A marked decrease in the saturation magnetization by the formation of functional shells around the magnetic core is an important disadvantage of magnetic core-shell nanoparticles. Another drawback of Ti(IV)-functionalized immobilized metal affinity chromatography (IMAC) sorbents is the acidic character of the binding medium used for Ti4+ attachment onto composite magnetic nanoparticles, which causes an additional decrease in the saturation magnetization owing to the chemical interaction between the acidic moiety and the magnetic core. An IMAC sorbent in the form of magnetic microspheres with superior and stable magnetic properties with respect to magnetic core-shell nanoparticles was designed for phosphopeptide enrichment. Magnetic, monodisperse-porous silica microspheres (MagSiO2) 6μm in size were synthesized by a new staged-shape template hydrolysis-condensation protocol. A porous-silica shell layer was generated around the microspheres to protect the magnetic core from the acidic medium during Ti4+ attachment (MagSiO2@SiO2). The MagSiO2@SiO2 microspheres were coated with a polydopamine shell (MagSiO2@SiO2@PDA) and Ti4+ was attached onto the composite microspheres (MagSiO2@SiO2@PDA@Ti(IV)). Formation of the PDA layer and Ti4+ attachment did not cause any significant decrease in the saturation magnetization. The platform exhibited excellent performance for phosphopeptide enrichment from the digests of phosphorylated proteins. Selectivity was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The detection limit for phosphopeptide enrichment by the MagSiO2@SiO2@PDA@Ti(IV) microspheres from the tryptic digests of β-casein was 50 fmol/mL. Usability of the proposed magnetic sorbent with complex biological samples was demonstrated by successful enrichment of four phosphopeptides from human serum. The proposed sorbent showed stable performance over five repeated uses. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry.

    Science.gov (United States)

    Sano, Akira; Nakamura, Hiroshi

    2004-05-01

    A method for the determination of phosphorylation sites in phosphoproteins based on column-switching high-performance liquid chromatography (HPLC) has been developed. The HPLC system consisted of a titania precolumn for the selective adsorption of phosphopeptides, an anion-exchange analytical column and a UV detector (215 nm). Rabbit muscle phosphorylase a (RPa) and porcine stomach pepsin (PSP) were tested as model phosphoproteins. After protease digestion, the resulting phosphopeptides were successfully isolated by column-switching HPLC. The phosphopeptide fractions were analyzed by electrospray ionization mass spectrometry with a positive or negative ion mode after purification by reversed-phase HPLC. Pseudo-molecular ion peaks corresponding to Gln-Ile-Ser(p)-Val-Arg (MW 681.7) and Glu-Ala-Thr-Ser(p)-Gln-Glu-Leu (MW 856.8) were detected from the tryptic digest of RPa and chymotryptic digest of PSP, respectively, which agreed with the theoretically expected phosphopeptide fragments.

  19. Phosphopeptide analysis of rodent epididymal spermatozoa.

    Science.gov (United States)

    Baker, Mark A; Hetherington, Louise; Weinberg, Anita; Velkov, Tony

    2014-12-30

    Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

  20. Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti(4+)-IMAC.

    Science.gov (United States)

    Piovesana, Susy; Capriotti, Anna Laura; Cavaliere, Chiara; Ferraris, Francesca; Samperi, Roberto; Ventura, Salvatore; Laganà, Aldo

    2016-02-25

    Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Synthesis of a new type of echinus-like Fe3O4@TiO2 core-shell-structured microspheres and their applications in selectively enriching phosphopeptides and removing phospholipids.

    Science.gov (United States)

    Li, Hua; Shi, Xianzhe; Qiao, Lizhen; Lu, Xin; Xu, Guowang

    2013-02-01

    Some compounds of low abundance in biological samples play important roles in bioprocesses. However, the detection of these compounds at inherently trace concentrations with interference from a complex matrix is difficult. New materials for sample pretreatment are essential for the removal of interferences and for selective enrichment. In this study, echinus-like Fe(3)O(4)@TiO(2) core-shell-structured microspheres (echinus-like microspheres) have been synthesized for the first time. Rutile phase TiO(2) nanorods with a length of approximately 300 nm and width of approximately 60 nm are arranged regularly on the surface of the microspheres. This novel type of material exhibited good selectivity and adsorption capacity toward phosphate-containing compounds. In proteomics research, the echinus-like microspheres were used to selectively enrich phosphopeptides from complex peptide mixtures. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis showed that fourteen phosphopeptides were detected from α-casein tryptic digests after enrichment. Even in peptide mixtures that contained highly abundant nonphosphorylated peptides with interference from bovine serum albumin, these phospopeptides could still be selectively trapped with little nonspecific adsorption. In metabolomics studies, the echinus-like microspheres were further used to selectively remove phosphocholines (PCs) and lysophosphocholines (LPCs), which are the main matrix interferences for the detection of metabolites of low abundance in plasma. Liquid chromatography-quadrupole time-of-flight mass spectrometry was used to perform the metabolic profiling of plasma. The high concentrations of PCs and LPCs were effectively eliminated, and many endogenous metabolites of low abundance were enhanced or even observed for the first time. All of the results suggest that echinus-like microspheres have potential applications in proteomics and metabolomics to improve the

  2. Phosphopeptide enrichment using MALDI plates modified with high-capacity polymer brushes.

    Science.gov (United States)

    Dunn, Jamie D; Igrisan, Elizabeth A; Palumbo, Amanda M; Reid, Gavin E; Bruening, Merlin L

    2008-08-01

    Matrix-assisted laser desorption/ionization plates coated with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes that are derivatized with Fe(III)-nitrilotriacetate (NTA) complexes allow selective, efficient phosphopeptide enrichment prior to analysis by mass spectrometry (MS). Fe(III)-NTA-PHEMA brushes (60 nm thick) have a phosphopeptide binding capacity of 0.6 microg/cm(2) and exhibit phosphopeptide recoveries of over 70%, whereas much thinner polymer films containing Fe(III)-NTA afford a recovery of only 20%, and a monolayer of Fe(III)-NTA shows a recovery of just 10%. Recoveries are determined by comparing signals from enriched unlabeled phosphopeptides with those of their deuterium-labeled analogues that were added to the plate just prior to addition of matrix. Mass spectra of phosphopeptide-containing samples enriched using Fe(III)-NTA-PHEMA-modified plates also demonstrate higher recoveries or fewer interfering peaks than corresponding spectra obtained with enrichment using several commercially available Fe(III)-containing films and resins or metal oxide materials. When analyzing tryptic digests of beta-casein, the Fe(III)-NTA-PHEMA brushes allow detection of as little as 15 fmol of phosphopeptide. Moreover, with both ovalbumin and beta-casein digests, phosphopeptide signals dominate the mass spectra obtained using these modified plates.

  3. Preparation of magnetic hydroxyapatite clusters and their application in the enrichment of phosphopeptides.

    Science.gov (United States)

    Yu, Qiao; Li, Xiao-Shui; Yuan, Bi-Feng; Feng, Yu-Qi

    2014-03-01

    A novel strategy for the effective enrichment of phosphopeptides based on magnetic hydro-xyapatite (HAp) clusters was developed in the current study. The structure of HAp ensures its probable separation capability, including cation exchange with P-sites (negatively charged pairs of crystal phosphates), calcium coordination, anion exchange with C-sites (positively charged pairs of crystal calcium ions). The prepared magnetic HAp clusters showed good performance on the efficient enrichment of phosphopeptides from the digestion mixture of β-casein and BSA. Compared to commercial HAp particles, the magnetic HAp clusters exhibited better selectivity toward phosphopeptides. In addition, the use of magnetic material greatly simplified the enrichment procedure, which avoided the tedious centrifugation steps in a typical phosphopeptides enrichment protocol. Finally, the material was successfully applied in the enrichment of phosphopeptides from human serum. Taken together, the efficient enrichment of the phosphopeptides by the easily prepared magnetic HAp clusters demonstrated a rapid and convenient strategy for the purification of phosphopeptides from complex samples, which may facilitate protein phosphorylation studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

    Science.gov (United States)

    Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

    2013-01-01

    A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O

  5. Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Chen, Cheng-Tai; Chen, Yu-Chie

    2005-09-15

    Columns packed with microsized titanium dioxide particles have been used effectively as precolumns for enriching phosphopeptides from complex mixtures. Nanosized titanium dioxide particles have a higher specific surface area and, hence, potentially higher trapping capacities toward phosphopeptides than do microsized particles. Thus, in this study, we employed TiO2-coated magnetic (Fe3O4/TiO2 core/shell) nanoparticles to selectively concentrate phosphopeptides from protein digest products. Because of their magnetic properties, the Fe3O4/TiO2 core/shell nanoparticles that are conjugated to the target peptides can be isolated readily from the sample solutions by employing a magnetic field. In this paper, we also demonstrate that the Fe3O4/TiO2 core/shell nanoparticles behave as an effective SALDI matrix: our upper detectable mass limit was approximately 24 000 Da, whereas the detection limit for peptides was in the low-femtomole range. That is to say, the target analytes trapped by the Fe3O4/TiO2 nanoparticles can be identified by introducing the particles directly into the mass spectrometer for TiO2-SALDI-MS analysis without the need for any further treatment. For example, elution steps are not necessary when using this approach. In addition, the trapping selectivity of these Fe3O4/TiO2 nanoparticles toward phosphopeptides was quite good. These properties combine to result in the low detection limits. The lowest detectable concentration of phosphopeptides that we analyzed using this approach was 500 pM for a 100-microL tryptic digest solution of beta-casein; this level is much lower than that which can be obtained using any other currently available method.

  6. Functionalized diamond nanopowder for phosphopeptides enrichment from complex biological fluids

    Energy Technology Data Exchange (ETDEWEB)

    Hussain, Dilshad [Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800 (Pakistan); Najam-ul-Haq, Muhammad, E-mail: najamulhaq@bzu.edu.pk [Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800 (Pakistan); Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 80-82, A-6020 Innsbruck (Austria); Jabeen, Fahmida; Ashiq, Muhammad N.; Athar, Muhammad [Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800 (Pakistan); Rainer, Matthias; Huck, Christian W.; Bonn, Guenther K. [Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 80-82, A-6020 Innsbruck (Austria)

    2013-05-02

    Graphical abstract: -- Highlights: •Derivatization of diamond nanopowder as IMAC and RP. •Characterization with SEM, EDX and FT-IR. •Phosphopeptide enrichment from standard as well as real samples. •Desalting and human serum profiling with reproducible results. •MALDI-MS analysis with database identification. -- Abstract: Diamond is known for its high affinity and biocompatibility towards biomolecules and is used exclusively in separation sciences and life science research. In present study, diamond nanopowder is derivatized as Immobilized Metal Ion Affinity Chromatographic (IMAC) material for the phosphopeptides enrichment and as Reversed Phase (C-18) media for the desalting of complex mixtures and human serum profiling through MALDI-TOF-MS. Functionalized diamond nanopowder is characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy. Diamond-IMAC is applied to the standard protein (β-casein), spiked human serum, egg yolk and non-fat milk for the phosphopeptides enrichment. Results show the selectivity of synthesized IMAC-diamond immobilized with Fe{sup 3+} and La{sup 3+} ions. To comprehend the elaborated use, diamond-IMAC is also applied to the serum samples from gall bladder carcinoma for the potential biomarkers. Database search is carried out by the Mascot program ( (www.matrixscience.com)) for the assignment of phosphorylation sites. Diamond nanopowder is thus a separation media with multifunctional use and can be applied to cancer protein profiling for the diagnosis and biomarker identification.

  7. Preparation of titanium-grafted magnetic mesoporous silica for the enrichment of endogenous serum phosphopeptides.

    Science.gov (United States)

    Li, Xiao-Shui; Pan, Ya-Ni; Zhao, Yong; Yuan, Bi-Feng; Guo, Lin; Feng, Yu-Qi

    2013-11-08

    As one of the most important post-translational modifications, reversible phosphorylation of protein plays crucial roles in a large number of biological processes. Moreover, endogenous phosphopeptides are also associated with certain human diseases. An efficient enrichment and separation method is the premise for successful identification and quantification of phosphopeptides. In this work, titanium grafted magnetic mesoporous silica (Fe3O4@Ti-mSiO2) was developed and applied for the enrichment of endogenous phosphopeptides. Fe3O4@Ti-mSiO2 particles were prepared by grafting titanocene dichloride on the inner walls of magnetic mesoporous silica and then being calcined to remove cyclopentadienyl ligand. The physicochemical properties of the prepared materials were characterized by energy dispersive X-ray spectroscopy (EDX), nitrogen adsorption-desorption analysis, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). For selective enrichment of phosphopeptides, the prepared Fe3O4@Ti-mSiO2 particles were applied for tryptic digests of β-casein, mixtures of β-casein and bovine serum albumin (BSA), and low-fat milk. Finally, Fe3O4@Ti-mSiO2 was successfully applied for the enrichment of endogenous phosphopeptides from human serum. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis.

    Science.gov (United States)

    Wu, Hsin-Yi; Tseng, Vincent Shin-Mu; Liao, Pao-Chi

    2007-05-01

    Protein phosphorylation is a key post-translational modification that governs biological processes. Despite the fact that a number of analytical strategies have been exploited for the characterization of protein phosphorylation, the identification of protein phosphorylation sites is still challenging. We proposed here an alternative approach to mine phosphopeptide signals generated from a mixture of proteins when liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is involved. The approach combined dephosphorylation reaction, accurate mass measurements from a quadrupole/time-of-flight mass spectrometer, and a computing algorithm to differentiate possible phosphopeptide signals obtained from the LC-MS analyses by taking advantage of the mass shift generated by alkaline phosphatase treatment. The retention times and m/z values of these selected LC-MS signals were used to facilitate subsequent LC-MS/MS experiments for phosphorylation site determination. Unlike commonly used neutral loss scan experiments for phosphopeptide detection, this strategy may not bias against tyrosine-phosphorylated peptides. We have demonstrated the applicability of this strategy to sequence more, in comparison with conventional data-dependent LC-MS/MS experiments, phosphopeptides in a mixture of alpha- and beta-caseins. The analytical scheme was applied to characterize the nasopharyngeal carcinoma (NPC) cellular phosphoproteome and yielded 221 distinct phosphorylation sites. Our data presented in this paper demonstrated the merits of computation in mining phosphopeptide signals from a complex mass spectrometric data set.

  9. Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N

    2009-01-01

    The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3+) and thi......The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3......+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensitive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working...

  10. [Selective medium to isolate human Bifidobacterium].

    Science.gov (United States)

    Huo, Dongxue; Zhang, Jiachao; Bai, Na; Huang, Weiqiang; Zhang, Heping

    2014-04-04

    To compare five selective media to isolate human Bifidobacterium. Feces from six healthy human volunteers were diluted and cultivated on five Bifidobacterium selective media. After anaerobic cultivation, bacterial colonies were counted, selected and identified. Meanwhile, bacterial genomic DNA was extracted from the feces samples, and the Denaturing Gradient Gel Electrophoresis (DGGE) and Quantitative Polymerase Chain Reaction (q-PCR) were applied to reveal the diversity of Bifidobacterium. The amount of Bifidobacterium grown on BSM and BLM media was similar to the result detected by q-PCR and was significantly higher than that on three other media. Bifidobacterium isolated from BLM medium was similar to the identified result of DGGE profile. BLM medium is the best selective medium for Bifidobacterium isolation from human gastrointestinal tract.

  11. Phosphopeptide Purification by IMAC with Fe(III) and Ga(III)

    DEFF Research Database (Denmark)

    Steen, Hanno; Stensballe, Allan

    2007-01-01

    of histidines. More recently, immobilized Fe(3+), Ga(3+), and Al(3+) metal ions have been used for the selective enrichment of phosphopeptides from complex proteolytic digest mixtures containing both phosphorylated and nonphosphorylated components. The use of a nitrilotriacetic acid (NTA) matrix over...

  12. Weaving a two-dimensional fishing net from titanoniobate nanosheets embedded with Fe3O4 nanocrystals for highly efficient capture and isotope labeling of phosphopeptides

    Science.gov (United States)

    Chen, Xueqin; Li, Siyuan; Zhang, Xiaoxia; Min, Qianhao; Zhu, Jun-Jie

    2015-03-01

    Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3O4 nanocrystals (Fe3O4-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe3O4 nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe3O4-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests.Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3

  13. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...

  14. Identification of a major phosphopeptide in human tristetraprolin by phosphopeptide mapping and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Heping Cao

    Full Text Available Tristetraprolin/zinc finger protein 36 (TTP/ZFP36 binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated in vivo and is a substrate for several protein kinases. Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS. Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83-103 of human TTP. MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP.

  15. The decreased of Streptococcus Mutans growth after topical application of phosphopeptide amorphous calcium phosphate paste

    Directory of Open Access Journals (Sweden)

    Tika Faradina Araf

    2011-07-01

    Full Text Available Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP paste is a topical application substance that consisted of a series of milk derivative peptide as a result of phosphorylation and has an antibacterial activity. The objective of this research was to find out the difference of Streptococcus mutans growth before and after CPP-ACP paste given topically to child's teeth. The method of the research was a quasi-experiment. Research samples were 10 students of MI Al Falah Islamic Boarding School, Jatinangor, West Jawa Indonesia and collected with purposive sampling technique. This research used dental plaque from child's teeth before and after applicated by CPP-ACP paste. The plaque was cultivated in selective media Tryptone Yeast Cysteine Sucrose Bacitracin (TYCSB with repeated twice. Streptococcus mutans colony in TYCSB were counted by Stuart colony counter and statistically analyzed based on paired t-test. The results showed the average of Streptococcus mutans growth before applicated CPP-ACP paste was 57.05, whereas after applicated CPP-ACP paste for 1 days was 9.4; for 3 days was 2.85, and for 14 days was 1.7 colony. The research concluded that there was a decrease of Streptococcus mutans growth in isolate plaque after CPP-ACP paste topically given to child's teeth.

  16. Design and synthesis of an immobilized metal affinity chromatography and metal oxide affinity chromatography hybrid material for improved phosphopeptide enrichment.

    Science.gov (United States)

    Yang, Da-Song; Ding, Xi-Ying; Min, Hong-Ping; Li, Bo; Su, Meng-Xiang; Niu, Miao-Miao; Di, Bin; Yan, Fang

    2017-07-07

    Reversible phosphorylation of proteins is one of the most important post-translational modifications, while the detection of phosphopeptides is difficult due to their low abundance and the signal suppression of nonphosphorylated peptides. Therefore, selective enrichment of phosphopeptides from highly complicated mixtures is vital for MS-based phosphoproteome analysis. Despite various strategies have been developed, there is no single method that is capable of providing full coverage of the whole phosphoproteome. Metal oxide affinity chromatography (MOAC) enrichment preferably singly phosphopeptides, whereas immobilized metal affinity chromatography (IMAC) enrichment bias towards multiply phosphopeptides. In this study, first example of IMAC and MOAC hybrid material, Fe3O4@nSiO2@mSiO2/TiO2-Ti4+ nanoparticles were successfully synthesized for the enrichment of phosphopeptides with the aim to combining their advantages for enrich both mono- and multi-phosphorylated species. The TiO2 was firstly coated on the surface of mesoporous silica and then grafted with 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) to chelate Ti4+ ions. This novel type of hybird material with high surface areas (179.3m2/g) exhibited good adsorption capacity (133mg/g) towards standard tryptic digest of β-casein and the method based on this material also showed good sensitivity (4pmol). The synthesized Fe3O4@nSiO2@mSiO2/TiO2-Ti4+ microspheres were further used to selectively enrich phosphopeptides from complex biosamples, seven mono-phosphopeptides and eight multi-phosphopeptides were successfully enriched from nonfat milk which is much better than single IMAC or MOAC strategy. Those results indicated that Fe3O4@nSiO2@mSiO2/TiO2-Ti4+ microspheres have potential applications in MS-based phosphoproteomics to enlarge phosphoproteomics coverage and this work was expected to open up a promising strategy which combined the advantages of various methods in one material for effective enrich

  17. Confident Site Localization Using a Simulated Phosphopeptide Spectral Library

    NARCIS (Netherlands)

    Suni, V.; Imanishi, S.Y.; Maiolica, A.; Aebersold, R.; Corthals, G.L.

    2015-01-01

    We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a

  18. Confident site localization using a simulated phosphopeptide spectral library.

    Science.gov (United States)

    Suni, Veronika; Imanishi, Susumu Y; Maiolica, Alessio; Aebersold, Ruedi; Corthals, Garry L

    2015-05-01

    We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.

  19. Isolation and selection of Bradyrhizobium from the root nodules of ...

    African Journals Online (AJOL)

    This research was conducted from 2004 to 2005 to isolate and select Bradyrhizobium from the root nodules of indigo plants (Indigofera tinctoria L.). For isolation of Bradyrhizobium, root nodules were collected from indigo plants. Fourteen Bradyrhizobium isolates were identified depending on colony, morphological and ...

  20. Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

    DEFF Research Database (Denmark)

    Ye, Juanying; Rudashevskaya, Elena; Hansen, Thomas Aarup

    stardand sample preparation protocols. Here, we combine 3 phosphpeptide enrichment methods (IMAC, TiO2 and Calcium Phosphate Precipitation (CPP)), and apply them to phosphoproteomic analysis of Arabidopsis thaliana plasma membrane preparation. Method Plant plasma membranes were isolated from Arabidopsis...... the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A...... (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany). Results We first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching...

  1. Hierarchically templated beads with tailored pore structure for phosphopeptide capture and phosphoproteomics

    DEFF Research Database (Denmark)

    Wierzbicka, Celina; Torsetnes, Silje B.; Jensen, Ole N.

    2017-01-01

    and crosslinkers inside the pores of macroporous silica beads with both free and immobilized template. In the final step the silica was removed by fluoride etching resulting in mesoporous polymer replicas with narrow pore size distributions, pore diameters ≈ 10 nm and surface area > 260 m2 g-1. The beads displayed...... pronounced phosphotyrosine affinity and selectivity in binding tests using model peptides in acetonitrile rich solutions with a performance surpassing solution polymerized bulk imprinted materials. Tests of the beads for the enrichment of phosphopeptides from tryptic digests of twelve proteins revealed both...

  2. Neighbor-directed histidine N(τ) alkylation. A route to imidazolium-containing phosphopeptide macrocycles

    Energy Technology Data Exchange (ETDEWEB)

    Qian, Wen-Jian [National Cancer Inst., Frederick, MD (United States); Park, Jung-Eun [National Cancer Inst., Bethesda, MD (United States); Grant, Robert [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Lai, Christopher C. [National Cancer Inst., Frederick, MD (United States); Kelley, James A. [National Cancer Inst., Frederick, MD (United States); Yaffe, Michael B. [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Lee, Kyung S. [National Cancer Inst., Bethesda, MD (United States); Burke, Terrence R. [National Cancer Inst., Frederick, MD (United States)

    2015-07-07

    Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. These cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Furthermore, neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.

  3. Effect of Fluoride, Casein Phosphopeptide-Amorphous Calcium Phosphate and Casein Phosphopeptide-Amorphous Calcium Phosphate Fluoride on Enamel Surface Microhardness After Microabrasion: An in Vitro Study.

    Science.gov (United States)

    Ahmadi Zenouz, Ghazaleh; Ezoji, Fariba; Enderami, Seyede Anese; Khafri, Soraya

    2015-10-01

    This study aimed to assess the effect of applying casein phosphopeptide- amorphous calcium phosphate (CPP-ACP) paste, casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) paste and sodium fluoride gel on surface microhardness of enamel after microabrasion. Thirty freshly extracted human premolars were selected. All samples were subjected to hardness indentations made with the Vickers hardness machine and the average value was recorded as the initial surface microhardness. The specimens were then randomly divided into three groups (n=10) of CPP-ACPF, fluoride and CPPACP. The teeth were micro-abraded with Opalustre. Microhardness test was performed to assess the post-abrasion hardness. Three remineralization modalities were performed on samples of each group. The enamel surface microhardness measurements were performed. To compare the difference between groups, the rehardening and softening values were defined. One-way ANOVA and Tukey's post hoc test at a significance level of 5% were used for statistical analysis. The mean microhardness value (MMV) had a significant decrease after microabrasion from baseline. The MMV had a significant increase after remineralization in all groups. The MMV of CPP-ACPF group was significantly more than that of fluoride group (P=0.027). The rehardening value of fluoride group was significantly more than that of other groups (Pmicroabrasion. The CPP-ACP and CPP-ACPF pastes are effective, but to a lesser extent than neutral sodium fluoride gel in remineralizing enamel surface. Incorporation of fluoride to CPP-ACP formulation does not provide any additional remineralizing potential.

  4. Selection and characterisation of monepantel resistance in Teladorsagia circumcincta isolates

    Directory of Open Access Journals (Sweden)

    D.J. Bartley

    2015-08-01

    To address these questions three MPTL-resistant Teladorsagia circumcincta isolates (MTci2-11, MTci5-13 and MTci7-12 have been artificially selected in vivo from phenotypically characterised parent isolates (MTci2, MTci5, MTci7 respectively. The selection process involved collecting and culturing eggs from surviving worms from sheep administered sub-optimal dosages of MPTL (Zolvix® to provide infective larvae to infect further sheep until resistant isolates were generated (between 9 and 13 rounds of selection. A controlled efficacy test was conducted using the original parental isolates and the newly generated MPTL resistant isolates (n = 5 per group. Selected isolates were assessed both under anthelmintic stress (Zolvix®, 2.5 mg/kg bodyweight; MTci-MPTL and at rest (untreated, MTci-CON. A number of life-history traits were assessed, namely, worm establishment rates, time to patency, faecal egg output, body length of adults and eggs in utero. The estimated resistance status of the selected isolates was confirmed with 48%, 28% and 9% reductions in worm burden at 7-days post Zolvix® administration for MTci2-11-MPTL, MTci5-13-MPTL and MTci7-12-MPTL, respectively, compared with untreated controls. One of the selected isolates MTci7-12-CON showed significantly greater total worm burden (p = 0.025, greater establishment rate (p = 0.033, decreased time to patency (p = 0.048, higher cumulative egg outputs (p = 0.002 compared with its parental derivative MTci7. The trial results suggest that anthelmintic selection in T. circumcincta, albeit under experimental conditions, can select for more prolific/fecund and quicker maturing populations. These data provide an insight into how parasites evolve in response to anthelmintic pressure.

  5. P189-T IMAC Enrichment of Phosphopeptides Coupled with 18O Labeling: Quantitative Phosphoproteomics

    Science.gov (United States)

    Turner, J. L.; Walters, J. J.; Heutel, J. D.; Radwanski, B. K.; Boland, J.; Ray, K.; Dapron, J. G.

    2007-01-01

    The up- and down-regulation of protein expression has long been a focus of proteomics researchers and has given rise to several mass spectrometry–based quantitation techniques. In the past decade, a great deal of attention has turned toward the subproteomes, particularly those representing post-translational modifications (PTMs), such as phosphorylation. Those PTMs often play key roles in signaling cascades, and affect a number of cellular processes; their study offers insight into intracellular function. We illustrate here phosphoproteome quantitation through the pairing of immobilized metal-affinity chromatography (IMAC) and 18O labeling, which allows for the determination of modulated phosphorylated species in response to external stressors. Following application of varying biological stressors and isolation of each resultant proteome, the samples were proteolytically cleaved using immobilized trypsin. IMAC enrichment of phosphorylated peptides was accomplished using a nitrilotriacetic acid (NTA) analog bonded to silica, presenting Ga3+ as the chelated metal species. Immobilized trypsin was also employed for incorporation of 18O into the sample for quantitative global labeling. It was determined that IMAC enrichment utilizing this combination of chelate, substrate, and metal provided significant enrichment of phosphopeptides that afforded signal levels, which allowed for quantitative analysis of the phosphoproteome. The use of 18O labeling of the samples permitted a global approach to targeting the phosphoproteome concurrent with the non-phosphorylated component. Using MS analysis before and after IMAC-based phosphopeptide enrichment revealed a distinction in regulation of expression and modulation through phosphorylation of the proteins. The coupling of phosphopeptide enrichment with global isotopic labeling provides a valuable and enabling resource for the vastly under-explored area of quantitative phosphoproteomics.

  6. Sensitivity of Colletotrichum acutatum Isolates to Selected Fungicides

    Directory of Open Access Journals (Sweden)

    Barbora Jílková

    2015-01-01

    Full Text Available Laboratory tests of six isolates of the pathogen Colletotrichum acutatum from different host plants demonstrated the varying sensitivity of pathogen with regard to mycelium growth and conidial germination after treatment with seven fungicides containing various active ingredients. None of the evaluated isolates was tolerant to the selected active ingredients in the fungicides. In tests of mycelium growth sensitivity, isolates from lupin and strawberry were most frequently identified as the most sensitive of all evaluated fungicides. The safflower isolate, on the other hand, most frequently exhibited the lowest reaction to fungicides. Differences in conidial germination of individual isolates were not detected in fungicides with the active ingredients dithianon, folpet and mancozeb, for which inhibition reached 100% in almost all isolates. The most significant differences in sensitivity among individual isolates were recorded in fungicides with the active ingredients azoxystrobin and metiram. In the case of the fungicide with active ingredient azoxystrobin, the highest inhibitory effect was achieved in the safflower isolate and the lowest in the white lupin isolate. After treatment with the fungicide with active ingredient metiram, the lowest germination rate was recorded in isolates from safflower and strawberry and the highest in isolates from hypericum and lupin.

  7. A novel tantalum-based sol-gel packed microextraction syringe for highly specific enrichment of phosphopeptides in MALDI-MS applications.

    Science.gov (United States)

    Çelikbıçak, Ömür; Atakay, Mehmet; Güler, Ülkü; Salih, Bekir

    2013-08-07

    A new tantalum-based sol-gel material was synthesized using a unique sol-gel synthesis pathway by PEG incorporation into the sol-gel structure without performing a calcination step. This improved its chemical and physical properties for the high capacity and selective enrichment of phosphopeptides from protein digests in complex biological media. The specificity of the tantalum-based sol-gel material for phosphopeptides was evaluated and compared with tantalum(V) oxide (Ta2O5) in different phosphopeptide enrichment applications. The tantalum-based sol-gel and tantalum(V) oxide were characterized in detail using FT-IR spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM), and also using a surface area and pore size analyzer. In the characterization studies, the surface morphology, pore volume, crystallinity of the materials and PEG incorporation into the sol-gel structure to produce a more hydrophilic material were successfully demonstrated. The X-ray diffractograms of the two different materials were compared and it was noted that the broad signals of the tantalum-based sol-gel clearly represented the amorphous structure of the sol-gel material, which was more likely to create enough surface area and to provide more accessible tantalum atoms for phosphopeptides to be easily adsorbed when compared with the neat and more crystalline structure of Ta2O5. Therefore, the phosphopeptide enrichment performance of the tantalum-based sol-gels was found to be remarkably higher than the more crystalline Ta2O5 in our studies. Phosphopeptides at femtomole levels could be selectively enriched using the tantalum-based sol-gel and detected with a higher signal-to-noise ratio by matrix-assisted laser desorption/ionization-mass spectrometer (MALDI-MS). Moreover, phosphopeptides in a tryptic digest of non-fat bovine milk as a complex real-world biological sample were retained with higher yield using a tantalum-based sol-gel. Additionally, the sol-gel material

  8. Calcium phosphopeptides -- mechanisms of action and evidence for clinical efficacy.

    Science.gov (United States)

    Cochrane, N J; Reynolds, E C

    2012-09-01

    Phosphoproteins/phosphopeptides with clusters of acidic residues are found throughout nature, where they aid in the prevention of unwanted precipitation of solid calcium phosphates. The acidic residues, particularly phosphoserine, interact with calcium and stabilize clusters of calcium and phosphate. Saliva and milk are two examples of biological fluids that contain such phosphoprotein/phosphopeptide-stabilized calcium phosphates, and both share a similar evolutionary pathway. Saliva has been shown to have remineralization potential and is of critical importance in maintaining the mineral content of teeth in the oral environment. Milk can be enzymatically modified to release casein phosphopeptides that contain the clusters of residues that allow milk to stabilize high concentrations of calcium and phosphate. These casein phosphopeptide-stabilized amorphous calcium phosphate nanocomplexes (CPP-ACP) can stabilize even higher concentrations of calcium and phosphate than milk and can be considered a salivary biomimetic, since they share many similarities to statherin. The mechanisms of action and the growing body of scientific evidence that supports the use of CPP-ACP to augment fluoride in inhibiting demineralization and enhancing the remineralization of white-spot lesions are reviewed.

  9. Selective isolation of mammalian genes by TAR cloning.

    Science.gov (United States)

    Kouprina, Natalay; Larionov, Vladimir

    2006-05-01

    Transformation-associated recombination (TAR) cloning provides a unique tool for selective isolation of desired chromosome segments and full-size genes from complex genomes in the form of a circular yeast artificial chromosome (YAC) up to 250 kb in size. The method has a broad application for structural and functional genomics, long-range haplotyping, mutational analysis of gene families, characterization of chromosomal rearrangements, and evolutionary studies. This unit describes a procedure for gene isolation by TAR as well as a method for conversion of YAC-TAR isolates into a bacterial artificial chromosome (BAC) form.

  10. Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays.

    Science.gov (United States)

    Panse, Sören; Dong, Liying; Burian, Antje; Carus, Robert; Schutkowski, Mike; Reimer, Ulf; Schneider-Mergener, Jens

    2004-01-01

    Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.

  11. Selection, isolation, and identification of fungi for bioherbicide production

    OpenAIRE

    Souza, Ang?lica Rossana Castro de; Baldoni, Daiana Bortoluzzi; Lima, Jessica; Porto, Vit?ria; Marcuz, Camila; Machado, Carolina; Ferraz, Rafael Camargo; Kuhn, Raquel C.; Jacques, Rodrigo J.S.; Guedes, Jerson V.C.; Mazutti, Marcio A.

    2016-01-01

    Abstract Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phyto...

  12. Microalgae Isolation and Selection for Prospective Biodiesel Production

    Directory of Open Access Journals (Sweden)

    Peer M. Schenk

    2012-06-01

    Full Text Available Biodiesel production from microalgae is being widely developed at different scales as a potential source of renewable energy with both economic and environmental benefits. Although many microalgae species have been identified and isolated for lipid production, there is currently no consensus as to which species provide the highest productivity. Different species are expected to function best at different aquatic, geographical and climatic conditions. In addition, other value-added products are now being considered for commercial production which necessitates the selection of the most capable algae strains suitable for multiple-product algae biorefineries. Here we present and review practical issues of several simple and robust methods for microalgae isolation and selection for traits that maybe most relevant for commercial biodiesel production. A combination of conventional and modern techniques is likely to be the most efficient route from isolation to large-scale cultivation.

  13. Highly selective enrichment of phosphorylated peptides using titanium dioxide

    DEFF Research Database (Denmark)

    Thingholm, Tine; Jørgensen, Thomas J D; Jensen, Ole N

    2006-01-01

    a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro...

  14. Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling

    Directory of Open Access Journals (Sweden)

    Benedetta Lombardi

    2015-03-01

    Full Text Available Defining alterations in signalling pathways in normal and malignant cells is becoming a major field in proteomics. A number of different approaches have been established to isolate, identify and quantify phosphorylated proteins and peptides. In the current report, a comparison between SCX prefractionation versus an antibody based approach, both coupled to TiO2 enrichment and applied to TMT labelled cellular lysates, is described. The antibody strategy was more complete for enriching phosphopeptides and allowed the identification of a large set of proteins known to be phosphorylated (715 protein groups with a minimum number of not previously known phosphorylated proteins (2.

  15. Selection of Infective Arbuscular Mycorrhizal Fungal Isolates for Field Inoculation

    Directory of Open Access Journals (Sweden)

    Elisa Pellegrino

    2010-09-01

    Full Text Available Arbuscular mycorrhizal (AM fungi play a key role in host plant growth and health, nutrient and water uptake, plant community diversity and dynamics. AM fungi differ in their symbiotic performance, which is the result of the interaction of two fungal characters, infectivity and efficiency. Infectivity is the ability of a fungal isolate to establish rapidly an extensive mycorrhizal symbiosis and is correlated with pre-symbiotic steps of fungal life cycle, such as spore germination and hyphal growth. Here, different AM fungal isolates were tested, with the aim of selecting infective endophytes for field inoculation. Greenhouse and microcosm experiments were performed in order to assess the ability of 12 AM fungal isolates to produce spores, colonize host roots and to perform initial steps of symbiosis establishment, such as spore germination and hyphal growth. AM fungal spore production and root colonization were significantly different among AM fungal isolates. Spore and sporocarp densities ranged from 0.8 to 7.4 and from 0.6 to 2.0 per gram of soil, respectively, whereas root colonization ranged from 2.9 to 72.2%. Percentage of spore or sporocarp germination ranged from 5.8 to 53.3% and hyphal length from 4.7 to 79.8 mm. The ordination analysis (Redundancy Analysis, RDA showed that environmental factors explained about 60% of the whole variance and their effect on fungal infectivity variables was significant (P = 0.002. The biplot clearly showed that variables which might be used to detect infective AM fungal isolates were hyphal length and root colonization. Such analysis may allow the detection of the best parameters to select efficient AM fungal isolates to be used in agriculture.

  16. Selection, isolation, and identification of fungi for bioherbicide production

    Directory of Open Access Journals (Sweden)

    Angélica Rossana Castro de Souza

    Full Text Available Abstract Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture.

  17. Selection, isolation, and identification of fungi for bioherbicide production.

    Science.gov (United States)

    Souza, Angélica Rossana Castro de; Baldoni, Daiana Bortoluzzi; Lima, Jessica; Porto, Vitória; Marcuz, Camila; Machado, Carolina; Ferraz, Rafael Camargo; Kuhn, Raquel C; Jacques, Rodrigo J S; Guedes, Jerson V C; Mazutti, Marcio A

    Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  18. Selection of cellulolytic fungi isolated from diverse substrates

    Directory of Open Access Journals (Sweden)

    Mônica Caramez Triches Damaso

    2012-08-01

    Full Text Available The aim of the present work was to select filamentous fungi isolated from diverse substrates to obtain the strains with potential to produce the hydrolytic enzymes. From a total of 215 strains, seven strains from the soils, six from the plants and one from sugarcane bagasse were selected and identified as belonging to the Trichoderma, Penicillium and Aspergillus genera. The best hydrolytic activities obtained by semi-solid fermentation using these strains were approximately: 35; 1; 160; 170 and 120 U/gdm (CMCase, FPase, β-glucosidase, xylanase and polygalacturonase, respectively, demonstrating their potential to synthesize the enzymes compared with the results reported in the literature.

  19. Chemical rationale for selection of isolates for genome sequencing

    DEFF Research Database (Denmark)

    Rank, Christian; Larsen, Thomas Ostenfeld; Frisvad, Jens Christian

    The advances in gene sequencing will in the near future enable researchers to affordably acquire the full genomes of handpicked isolates. We here present a method to evaluate the chemical potential of an entire species and select representatives for genome sequencing. The selection criteria for new...... strains to be sequenced can be manifold, but for studying the functional phenotype, using a metabolome based approach offers a cheap and rapid assessment of critical strains to cover the chemical diversity. We have applied this methodology on the complex A. flavus/A. oryzae group. Though these two species...... are in principal identical, they represent two different phenotypes. This is clearly presented through a correspondence analysis of selected extrolites, in which the subtle chemical differences are visually dispersed. The results points to a handful of strains, which, if sequenced, will likely enhance our...

  20. On-bead chemical synthesis and display of phosphopeptides for affinity pull-down proteomics

    DEFF Research Database (Denmark)

    Malene, Brandt; Madsen, Jens C.; Bunkenborg, Jakob

    2006-01-01

    (aldehyde) at the C terminus for potential activity-based proteomics. The synthetic support-bound Bad phosphopeptides were able to pull down 14-3-3zeta. Furthermore, Bad phosphopeptides bound endogenous 14-3-3 proteins, and all seven members of the 14-3-3 family were identified by mass spectrometry......We describe a new method for phosphopeptide proteomics based on the solid-phase synthesis of phosphopeptides on beads suitable for affinity pull-down experiments. Peptide sequences containing the Bad Ser112 and Ser136 phosphorylation motifs were used as bait in affinity pull-down experiments....... In control experiments, none of the unphosphorylated Bad peptides bound transfected 14-3-3zeta or endogenous 14-3-3. We conclude that the combined synthesis and display of phosphopeptides on-bead is a fast and efficient method for affinity pull-down proteomics....

  1. Neighbor-directed Histidine N(τ)–Alkylation: A Route to Imidazolium-containing Phosphopeptide Macrocycles

    Science.gov (United States)

    Qian, Wen-Jian; Park, Jung-Eun; Grant, Robert; Lai, Christopher C.; Kelley, James A.; Yaffe, Michael B.; Lee, Kyung S.; Burke, Terrence R.

    2016-01-01

    Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In our current work, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts. PMID:26152807

  2. Neighbor-Directed Histidine N (s)–Alkylation: A Route to Imidazolium-Containing Phosphopeptide Macrocycles-Biopolymers | Center for Cancer Research

    Science.gov (United States)

    Our recently discovered, selective, on-resin route to N(s)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation.

  3. Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

    Science.gov (United States)

    Beck, A; Deeg, M; Moeschel, K; Schmidt, E K; Schleicher, E D; Voelter, W; Häring, H U; Lehmann, R

    2001-01-01

    A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis. Copyright 2001 John Wiley & Sons, Ltd.

  4. Facile Synthesis of Mesocrystalline SnO2 Nanorods on Reduced Graphene Oxide Sheets: An Appealing Multifunctional Affinity Probe for Sequential Enrichment of Endogenous Peptides and Phosphopeptides.

    Science.gov (United States)

    Ma, Wen; Zhang, Feng; Li, Liping; Chen, Shuai; Qi, Limin; Liu, Huwei; Bai, Yu

    2016-12-28

    A novel multifunctional composite comprising mesocrystalline SnO2 nanorods (NRs) vertically aligned on reduced graphene oxide (rGO) sheets was synthesized and developed for sequential capture of endogenous peptides and phosphopeptides. With the hydrophobicity of rGO and high affinity of SnO2 nanorods, sequential enrichment of endogenous peptides and phosphopeptides could be easily achieved through a modulation of elution buffer. With this multifunctional nanomaterial, 36 peptides were observed from diluted bovine serum albumin (BSA) tryptic digest and 4 phosphopeptides could be selectively captured from β-casein digest. The detection limit of tryptic digest of β-casein was low to 4 × 10(-10) M, and the selectivity was up to 1:500 (molar ratio of β-casein and BSA digest). The effectiveness and robustness of rGO-SnO2 NRs in a complex biological system was also confirmed by using human serum as a real sample. Our work is promising for small peptide enrichment and identification especially in complicated biological sample preparation, which also opens a new perspective in the design of multifunctional affinity probes for proteome or peptidome.

  5. Interactions of selected bacterial isolates with DBT and solubilized coal

    Energy Technology Data Exchange (ETDEWEB)

    Key, D.H.; Fox, R.V.; Kase, R.S.; Willey, M.S.; Stoner, D.L.; Ward, T.E.

    1990-01-01

    We are studying the interactions of isolated bacteria with dibenzothiophene (DBT), a sulfur-containing model compound, and with a solubilized coal product derived from a high-organic-sulfur lignite. The sensitivity of the tetrazolium assay used to identify and study these strains was improved by substituting tetrazolium violet for triphenyltetrazolium. DBT metabolism by thirteen strains was investigated using qualitative and quantitative GC and GC-MS analyses. Growth medium and incubation time affect the extent of DBT degradation and the production of DBT metabolites. Under specific conditions, seven of the strains produce metabolites which elute close to the position of one or another of the biphenyl standards. However, when these samples are spiked with the standard compounds, the bacterial metabolites do not co-elute with the standards. The modification of solubilized high-organic-sulfur coal by six of these strains was also studied. No selective removal of sulfur relative to carbon was observed. 13 refs., 1 fig., 2 tabs.

  6. Phosphopeptide characterization by mass spectrometry using reversed-phase supports for solid-phase β-elimination/Michael addition.

    Science.gov (United States)

    Nika, Heinz; Lee, JaeHoon; Willis, Ian M; Angeletti, Ruth Hogue; Hawke, David H

    2012-07-01

    We have adapted the Ba(2+) ion-catalyzed concurrent Michael addition reaction to solid-phase derivatization on ZipTip(C18) pipette tips using 2-aminoethanethiol as a nucleophile. This approach provides several advantages over the classical in-solution-based techniques, including ease of operation, completeness of reaction, improved throughput, efficient use of dilute samples, and amenability to automation. Phosphoseryl and phosphothreonyl peptides, as well as phosphoserine peptides with adjoining prolines, were used to optimize the reaction conditions, which proved highly compatible with the integrity of the samples. The analyte was recovered from the silica-based C18 resin at minimal sample loss. The use of the protocol for improved phosphopeptide detection by signal enhancement was demonstrated with low-level amounts of proteolytic digests from model proteins and experimental samples, an effect found especially prominent with multiple phosphorylated species. The reaction products proved highly suitable for structural characterization by collisionally induced dissociation (CID), and the resultant increased spectral information content, greatly facilitating mapping of the site of phosphorylation. In select cases, the method enables phosphorylation site localization within known protein sequences on the basis of single-stage data alone. The solid-phase strategy presented here provides a simple, versatile, and efficient tool for phosphopeptide structural characterization equipment readily available in most biological laboratories.

  7. Stimulus Selection for Intranasal Sensory Isolation: Eugenol Is an Irritant

    Science.gov (United States)

    Wise, Paul M.; Lundström, Johan N.

    2012-01-01

    Both the olfactory and the trigeminal systems are able to respond to intranasal presentations of chemical vapor. Accordingly, when the nose detects a volatile chemical, it is often unclear whether we smell it, feel it, or both. The distinction may often be unimportant in our everyday perception of fragrances or aromas, but it can matter in experiments that purport to isolate olfactory processes or study the interaction between olfaction and chemesthesis. Researchers turn to a small pool of compounds that are believed to be “pure olfactory” stimuli with little or no trigeminal impact. The current report reexamines one such commonly used compound, namely eugenol, a flavor and fragrance ingredient that has anesthetic properties under some conditions. Using a standard method involving many trials during an experimental session (Experiment 1), subjects were unable to reliably lateralize eugenol, consistent with claims that this compound is detected primarily through olfaction. However, with more limited exposure (Experiments 2 and 3), subjects were able to lateralize eugenol. We speculate that anesthetic properties of eugenol could blunt its trigeminal impact in some paradigms. Regardless, the current experiments suggest that eugenol can in fact stimulate the trigeminal nerve but in a complex concentration–dependent manner. Implications and strategies for selection of model odorants are discussed. PMID:22293937

  8. Molecular characterization of rotavirus isolates from select Canadian pediatric hospitals

    Directory of Open Access Journals (Sweden)

    McDermid Andrew

    2012-11-01

    Full Text Available Abstract Background We report the first multi-site rotavirus genotype analysis in Canada. Prior to this study, there was a dearth of rotavirus G and P genotyping data in Canada. Publically funded universal rotavirus vaccination in Canada started in 2011 and has been introduced by four provinces to date. Uptake of rotavirus vaccines in Canada prior to 2012 has been very limited. The aim of this study was to describe the genotypes of rotavirus strains circulating in Canada prior to widespread implementation of rotavirus vaccine by genotyping samples collected from selected paediatric hospitals. Secondly we identified rotavirus strains that differed genetically from those included in the vaccines and which could affect vaccine effectiveness. Methods Stool specimens were collected by opportunity sampling of children with gastroenteritis who presented to emergency departments. Samples were genotyped for G (VP7 genotypes and P (VP4 genotypes by hemi-nested multiplex PCR methods. Phylogenetic analysis was carried out on Canadian G9 strains to investigate their relationship to G9 strains that have circulated in other regions of the world. Results 348 samples were collected, of which 259 samples were rotavirus positive and genotyped. There were 34 rotavirus antigen immunoassay negative samples genotyped using PCR-based methods. Over the four rotavirus seasons, 174 samples were G1P[8], 45 were G3P[8], 22 were G2P[4], 13 were G9P[8], 3 were G4P[8] and 2 were G9P[4]. Sequence analysis showed that all Canadian G9 isolates are within lineage III. Conclusions Although a limited number of samples were obtained from a median of 4 centres during the 4 years of the study, it appears that currently approved rotavirus vaccines are well matched to the rotavirus genotypes identified at these hospitals. Further surveillance to monitor the emergence of rotavirus genotypes in Canada is warranted.

  9. Molecular characterization of rotavirus isolates from select Canadian pediatric hospitals

    Science.gov (United States)

    2012-01-01

    Background We report the first multi-site rotavirus genotype analysis in Canada. Prior to this study, there was a dearth of rotavirus G and P genotyping data in Canada. Publically funded universal rotavirus vaccination in Canada started in 2011 and has been introduced by four provinces to date. Uptake of rotavirus vaccines in Canada prior to 2012 has been very limited. The aim of this study was to describe the genotypes of rotavirus strains circulating in Canada prior to widespread implementation of rotavirus vaccine by genotyping samples collected from selected paediatric hospitals. Secondly we identified rotavirus strains that differed genetically from those included in the vaccines and which could affect vaccine effectiveness. Methods Stool specimens were collected by opportunity sampling of children with gastroenteritis who presented to emergency departments. Samples were genotyped for G (VP7) genotypes and P (VP4) genotypes by hemi-nested multiplex PCR methods. Phylogenetic analysis was carried out on Canadian G9 strains to investigate their relationship to G9 strains that have circulated in other regions of the world. Results 348 samples were collected, of which 259 samples were rotavirus positive and genotyped. There were 34 rotavirus antigen immunoassay negative samples genotyped using PCR-based methods. Over the four rotavirus seasons, 174 samples were G1P[8], 45 were G3P[8], 22 were G2P[4], 13 were G9P[8], 3 were G4P[8] and 2 were G9P[4]. Sequence analysis showed that all Canadian G9 isolates are within lineage III. Conclusions Although a limited number of samples were obtained from a median of 4 centres during the 4 years of the study, it appears that currently approved rotavirus vaccines are well matched to the rotavirus genotypes identified at these hospitals. Further surveillance to monitor the emergence of rotavirus genotypes in Canada is warranted. PMID:23153184

  10. Production of Microbial Protease from Selected Soil Fungal Isolates

    African Journals Online (AJOL)

    Dr Oseni

    establishing the industrial and biotechnological importance of this microbial enzyme. The harvested mycelia of the fungi ... Keywords: Soil microorganism, fungal isolate, incubation period, microbial enzyme. Correspondence: ..... highlands, Sarawak. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC).

  11. Comparison of Four Selective Agars for the Isolation of Pseudomonads

    Science.gov (United States)

    Hart, A.; Kite, Atricia E.

    1977-01-01

    Significant differences were found between Dettol (chloroxylenol) agar, nalidixic acid-cetrimide agar, and two other cetrimide-containing agars used for the isolation of small numbers of pseudomonads. PMID:879777

  12. Influence of selected Rhizoctonia solani isolates on sugar beet seedlings

    Directory of Open Access Journals (Sweden)

    Skonieczek Paweł

    2016-04-01

    Full Text Available From 2008 to 2010 the levels of sugar beet seedlings infection caused by Rhizoctonia solani were compared in laboratory tests. Seven sugar beet lines were tested: H56, H66, S2, S3, S4, S5 and S6 as well as three control cultivars: Carlos, Esperanza and Janosik. Sugar beet lines with tolerance to rhizoctoniosis and cultivars without tolerance were infected artificially by R. solani isolates: R1, R28a and R28b. These isolates belong to the second anastomosis group (AG, which is usually highly pathogenic to beet roots. The aim of the experiment was to test whether the tolerance of sugar beet genotypes to R. solani AG 2 prevents both root rot, and damping-off of seedlings, induced by the pathogen. Sugar beet lines tolerant to brown root rot in laboratory tests were significantly less sensitive to infection of the seedlings by R. solani AG 2 isolates in comparison to control cultivars. Rhizoctonia solani AG 2 isolates demonstrated considerable differences in pathogenicity against seedlings of sugar beet lines and cultivars. The strongest infection of sugar beet seedlings occurred with the isolate R28b. The greatest tolerance to infection by AG 2 isolates was found for the S5 and S3 breeding lines.

  13. Comparison of four selective media for the isolation of Burkholderia mallei and Burkholderia pseudomallei.

    Science.gov (United States)

    Glass, Mindy B; Beesley, Cari A; Wilkins, Patricia P; Hoffmaster, Alex R

    2009-06-01

    Currently there are no commercially available selective media indicated for the isolation of Burkholderia mallei and Burkholderia pseudomallei. Ashdown's agar, a custom selective medium for isolation of B. pseudomallei, is well described in the literature but unavailable commercially. Three commercially available media, Burkholderia cepacia selective agar (BCSA), oxidative-fermentative-polymyxin B-bacitracin-lactose (OFPBL) agar, and Pseudomonas cepacia (PC) agar are recommended for isolation of B. cepacia from respiratory secretions of cystic fibrosis patients. We evaluated the sensitivity and selectivity of these four media using 20 B. mallei, 20 B. pseudomallei, 20 Burkholderia spp., and 15 diagnostically challenging organisms. Ashdown's agar was the most sensitive medium for the isolation of B. pseudomallei, but it was unable to support growth of B. mallei. Pseudomonas cepacia agar was highly sensitive and selective for both organisms. In non-endemic areas, we suggest the use of the commercially available PC agar for the isolation of B. mallei and B. pseudomallei.

  14. Selection for autochthonous bifidobacteial isolates adapted to simulated gastrointestinal fluid

    Directory of Open Access Journals (Sweden)

    H Jamalifar

    2010-03-01

    Full Text Available "nBackground and the purpose of the study: Bifidobacterial strains are excessively sensitive to acidic conditions and this can affect their living ability in the stomach and fermented foods, and as a result, restrict their use as live probiotic cultures. The aim of the present study was to obtain bifidobacterial isolates with augmented tolerance to simulated gastrointestinal condition using cross-protection method. "nMethods: Individual bifidobacterial strains were treated in acidic environment and also in media containing bile salts and NaCl. Viability of the acid and acid-bile-NaCl tolerant isolates was further examined in simulated gastric and small intestine by subsequent incubation of the probiotic bacteria in the corresponding media for 120 min. Antipathogenic activities of the adapted isolates were compared with those of the original strains. "nResults and major conclusion: The acid and acid-bile-NaCl adapted isolates showed improved viabilities significantly (p<0.05 in simulated gastric fluid compared to their parent strains. The levels of reduction in bacterial count (Log cfu/ml of the acid and acid-bile-NaCl adapted isolates obtained in simulated gastric fluid ranged from 0.64-3.06 and 0.36-2.43 logarithmic units after 120 min of incubation. There was no significant difference between the viability of the acid-bile-NaCl-tolerant isolates and the original strains in simulated small intestinal condition except for Bifidobacterium adolescentis (p<0.05. The presence of 15 ml of supernatants of acid-bile-NaCl-adapted isolates and also those of the initial Bifidobacterium strains inhibited pathogenic bacterial growth for 24 hrs. Probiotic bacteria with improved ability to survive in harsh gastrointestinal environment could be obtained by subsequent treatment of the strains in acid, bile salts and NaCl environments.

  15. Isolation, optimization and characterization of selected Cyanophycean members

    Digital Repository Service at National Institute of Oceanography (India)

    Nagle, V.L.; Mhalsekar, N.M.; Jagtap, T.G.

    , and the subsequent purification. The techniques normally used to isolate the algal species, which could readily be cultured1. Various approaches have been adopted to develop efficient methods to isolate culture and purify fresh water BGA2-7. Efforts also have... reported to be poor in lipids. Spirulina contained only 6-13% lipids, half of which were fatty acids24,26 & 27. Present results were also revealed low concentrations of lipids in BGA. Salinity affects living organisms by decreasing the water...

  16. Manipulating the fragmentation patterns of phosphopeptides via gas-phase boron derivatization: determining phosphorylation sites in peptides with multiple serines.

    Science.gov (United States)

    Gronert, Scott; Li, Kathy H; Horiuchi, Mizue

    2005-12-01

    Trivalent boron species readily react with protonated phosphopeptides to give addition products with the loss of boron ligands. In the present study, trimethoxyborane (TMB), diisopropoxymethylborane (DIPM), and diethylmethoxyborane (DEMB) were allowed to react with four phosphopeptides, VsSF, LSsF, LsGASA, and VSGAsA (lower-case s indicates phosphoserine). Each of the phosphopeptides contains one serine that is phosphorylated and one that is not. Under collision-activated dissociation (CAD) conditions, the boron-derivatized peptides give fragmentation patterns that differ significantly from that of the protonated phosphopeptide. The patterns vary, depending on the number of labile (i.e., alkoxy) ligands on the boron. In general, boron derivatization increases the yield of phosphate-containing sequence ions, but dramatic effects are only seen with certain reagent/peptide combinations. However, the suite of reagents provides a means of altering and increasing the information content of phosphopeptide CAD spectra.

  17. In Vitro Susceptibility of Mycobacterium ulcerans Isolates to Selected Antimicrobials

    Directory of Open Access Journals (Sweden)

    Enid Owusu

    2017-01-01

    Full Text Available Background. The current definitive treatment of Buruli ulcer with antibiotics makes the issue of antimicrobial drug resistance an unavoidable one. This is as a result of drug misuse by health personnel and patients’ noncompliance to treatment regimen. Monitoring of these factors and screening for new effective antimicrobials are crucial to effective management of Buruli ulcer disease. This study therefore investigated the inhibitory activity of some antibiotics against isolates of Mycobacterium ulcerans. Methods. Activity of eight antibiotics was tested against twelve M. ulcerans isolates (2 reference strains and 10 clinical isolates. The anti-M. ulcerans activities were determined by the agar dilution method and the minimum inhibitory concentrations (MICs were determined by the agar proportion method. Results. All antimicrobials investigated had activity against M. ulcerans isolates tested. The MICs ranged from 0.16 μg/mL to 2.5 μg/mL. Azithromycin recorded the highest inhibitory activity at a mean MIC of 0.39 μg/mL, whilst clofazimine a second-line antileprosy drug, recorded the lowest at a mean MIC of 2.19 μg/mL. Among the four antituberculosis drugs, rifampicin had the highest activity with a mean MIC of 0.81 μg/mL. Conclusion. Azithromycin could be considered as a lucrative alternative to existing treatment methods for inhibiting M. ulcerans in Ghana.

  18. Antibacterial Activities of Lactic Acid Bacteria Isolated from Selected ...

    African Journals Online (AJOL)

    Members of lactic acid bacteria (LAB) are known probiotics and have been reported to have antimicrobial properties. Although various researchers have documented the isolation of these bacteria from fruits and vegetables, studies on LAB associated with lettuce, cucumber and cabbage are limited and non-existing in ...

  19. evaluation of the spoilage potential of selected bacterial isolates on ...

    African Journals Online (AJOL)

    BACTERIAL ISOLATES ON EFHIOPIAN SAUCES AND. EFFECT OF TWO ... The ingredients of the various spices may also affect the extent of proliferation of .... of growth was seen in “berbere” medium than in control in four strains. (p>0.05).

  20. Production of Microbial Protease from Selected Soil Fungal Isolates ...

    African Journals Online (AJOL)

    This study was undertaken to monitor the production of protease enzyme from soil fungal isolates obtained from Omo natural forest in Ogun State of Nigeria. The study also sought to determine the kinetic parameters of the enzyme with the aim of establishing the industrial and biotechnological importance of this microbial ...

  1. Characterization of Novel Trichoderma asperellum Isolates to Select Effective Biocontrol Agents Against Tomato Fusarium Wilt

    Directory of Open Access Journals (Sweden)

    Mahmoud H. El_Komy

    2015-03-01

    Full Text Available The use of novel isolates of Trichoderma with efficient antagonistic capacity against Fusarium oxysporum f. sp. lycopersici (FOL is a promising alternative strategy to pesticides for tomato wilt management. We evaluated the antagonistic activity of 30 isolates of T. asperellum against 4 different isolates of FOL. The production of extracellular cell wall degrading enzymes of the antagonistic isolates was also measured. The random amplified polymorphic DNA (RAPD method was applied to assess the genetic variability among the T. asperellum isolates. All of the T. asperellum isolates significantly reduced the mycelial growth of FOL isolates but the amount of growth reduction varied significantly as well. There was a correlation between the antagonistic capacity of T. asperellum isolates towards FOL and their lytic enzyme production. Isolates showing high levels of chitinase and β-1,3-glucanase activities strongly inhibited the growth of FOL isolates. RAPD analysis showed a high level of genetic variation among T. asperellum isolates. The UPGMA dendrogram revealed that T. asperellum isolates could not be grouped by their anta- gonistic behavior or lytic enzymes production. Six isolates of T. asperellum were highly antagonistic towards FOL and potentially could be used in commercial agriculture to control tomato wilt. Our results are consistent with the conclusion that understanding the genetic variation within Trichoderma isolates and their biochemical capabilities are required for the selection of effective indigenous fungal strains for the use as biocontrol agents.

  2. Evaluation of three selective media for isolation of Aggregatibacter actinomycetemcomitans

    NARCIS (Netherlands)

    Rurenga, P; Raangs, E; Singadji, Z; Wekema - Mulder, G; Veloo, A C M; van Winkelhoff, A J

    2013-01-01

    Background and Objective: Aggregatibacter actinomycetemcomitans is a pathogen in oral and nonoral infections. Detection and quantification of this pathogen can be performed using selective culture techniques. The aim of this study was to establish the efficacy of two known selective media in their

  3. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

    DEFF Research Database (Denmark)

    Kjellström, Sven; Jensen, Ole Nørregaard

    2004-01-01

    ,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins alpha-casein and beta-casein. The beneficial effects of adding up to 1% phosphoric...... acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix....

  4. [Isolation of Actinomycetales from the soil of Kazakhstan on selective media with antibiotics].

    Science.gov (United States)

    Vetlugina, L A; Adiiatova, Zh F; Khozhamuratova, S Sh; Rymzhanova, Z A; Trenozhnikova, L P; Kopytina, M N

    1990-02-01

    About 3000 actinomycetes were isolated from various soil samples collected in 11 regions of Kazakhstan. 62.7 per cent of them proved to be antagonists. For isolation of the strains, selective media supplemented with antibiotics were used. Kanamycin promoted growth of Actinomadura and Nocardia. Rubomycin promoted growth of Actinomadura. Tavromycetin and roseofungin were used as selective agents for the first time. Tavromycetin favoured isolation of Actinomadura and Nocardia. Roseofungin favoured isolation of Actinomadura. Light chestnut and serozemic soils were the most rich in antagonists (67.1 and 61.3 per cent, respectively) while saline and chestnut soils were the poorest in antagonists (32.2 and 30.6 per cent, respectively). Actinomadura were more frequent in light-chestnut light-loamy and serozemic soils. Half of the antibiotics isolated in the form of concentrates were identified with the known antibiotics or classified as belonging to various groups. A culture producing a novel antibiotic was isolated.

  5. Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (Araucaria angustifolia)

    National Research Council Canada - National Science Library

    Ribeiro, Carlos Marcelo; Cardoso, Elke Jurandy Bran Nogueira

    2012-01-01

    ... been submitted to intense predatory exploitation during the last century. Root-associated bacteria from A. angustifolia were isolated, selected and characterized for their biotechnological potential of growth promotion and biocontrol of plant pathogenic fungi...

  6. An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater

    Science.gov (United States)

    Kim, Jinyong; Oh, Euna; Banting, Graham S.; Braithwaite, Shannon; Chui, Linda; Ashbolt, Nicholas J.; Neumann, Norman F.; Jeon, Byeonghwa

    2016-01-01

    Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin), to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with Ct value using quantitative real-time PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater. PMID:27617011

  7. Effective Enrichment and Mass Spectrometry Analysis of Phosphopeptides Using Mesoporous Metal Oxide Nanomaterials

    Science.gov (United States)

    Nelson, Cory A.; Szczech, Jeannine R.; Dooley, Chad J.; Xu, Qingge; Lawrence, Matthew J.; Zhu, Haoyue; Jin, Song; Ge, Ying

    2010-01-01

    Mass spectrometry (MS)-based phosphoproteomics remains challenging due to the low abundance of phosphoproteins and substoichiometric phosphorylation. This demands better methods to effectively enrich phosphoproteins/peptides prior to MS analysis. We have previously communicated the first use of mesoporous zirconium oxide (ZrO2) nanomaterials for effective phosphopeptide enrichment. Here we present the full report including the synthesis, characterization, and application of mesoporous titanium dioxide (TiO2), ZrO2, and hafnium oxide (HfO2) in phosphopeptide enrichment and MS analysis. Mesoporous ZrO2 and HfO2 are demonstrated to be superior to TiO2 for phosphopeptide enrichment from a complex mixture with high specificity (>99%), which could almost be considered as “a purification”, mainly because of the extremely large active surface area of mesoporous nanomaterials. A single enrichment and Fourier transform MS analysis of phosphopeptides digested from a complex mixture containing 7% of α-casein identified 21 out of 22 phosphorylation sites for α-casein. Moreover, the mesoporous ZrO2 and HfO2 can be reused after a simple solution regeneration procedure with comparable enrichment performance to that of fresh materials. Mesoporous ZrO2 and HfO2 nanomaterials hold great promise for applications in MS-based phosphoproteomics. PMID:20704311

  8. Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples

    DEFF Research Database (Denmark)

    Ye, Juanying; Zhang, Xumin; Young, Clifford

    2010-01-01

    peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed...

  9. Uranyl Photocleavage of Phosphopeptides Yields Truncated C-Terminally Amidated Peptide Products

    DEFF Research Database (Denmark)

    Elnegaard, Rasmus L B; Møllegaard, Niels Erik; Zhang, Qiang

    2017-01-01

    The uranyl ion (UO2(2+) ) binds phosphopeptides with high affinity, and when irradiated with UV-light, it can cleave the peptide backbone. In this study, high-accuracy tandem mass spectrometry and enzymatic assays were used to characterise the photocleavage products resulting from the uranyl phot...

  10. Improved selective medium for the isolation of Treponema hyodysenteriae.

    OpenAIRE

    Kunkle, R A; Kinyon, J M

    1988-01-01

    An agar medium with improved selection for Treponema hyodysenteriae was developed. Cultures of T. hyodysenteriae and T. innocens, feces from 11 clinically normal pigs, and colonic contents from 6 pigs with gross lesions consistent with swine dysentery were diluted in phosphate-buffered saline and plated on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 5% citrated bovine blood (TSA), TSA with 400 micrograms of spectinomycin per ml (TSA-S400), TSA-S400 with 25 microgram...

  11. Contributions of natural and sexual selection to the evolution of premating reproductive isolation: a research agenda.

    Science.gov (United States)

    Safran, Rebecca J; Scordato, Elizabeth S C; Symes, Laurel B; Rodríguez, Rafael L; Mendelson, Tamra C

    2013-11-01

    Speciation by divergent natural selection is well supported. However, the role of sexual selection in speciation is less well understood due to disagreement about whether sexual selection is a mechanism of evolution separate from natural selection, as well as confusion about various models and tests of sexual selection. Here, we outline how sexual selection and natural selection are different mechanisms of evolutionary change, and suggest that this distinction is critical when analyzing the role of sexual selection in speciation. Furthermore, we clarify models of sexual selection with respect to their interaction with ecology and natural selection. In doing so, we outline a research agenda for testing hypotheses about the relative significance of divergent sexual and natural selection in the evolution of reproductive isolation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. The accumulation of reproductive isolation in early stages of divergence supports a role for sexual selection.

    Science.gov (United States)

    Martin, M D; Mendelson, T C

    2016-04-01

    Models of speciation by sexual selection propose that male-female coevolution leads to the rapid evolution of behavioural reproductive isolation. Here, we compare the strength of behavioural isolation to ecological isolation, gametic incompatibility and hybrid inviability in a group of dichromatic stream fishes. In addition, we examine whether any of these individual barriers, or a combined measure of total isolation, is predicted by body shape differences, male colour differences, environmental differences or genetic distance. Behavioural isolation reaches the highest values of any barrier and is significantly greater than ecological isolation. No individual reproductive barrier is associated with any of the predictor variables. However, marginally significant relationships between male colour and body shape differences with ecological and behavioural isolation are discussed. Differences in male colour and body shape predict total reproductive isolation between species; hierarchical partitioning of these two variables' effects suggests a stronger role for male colour differences. Together, these results suggest an important role for divergent sexual selection in darter speciation but raise new questions about the mechanisms of sexual selection at play and the role of male nuptial ornaments. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

  13. Comparative evaluation of Nano-hydroxyapatite and casein Phosphopeptide-amorphous calcium phosphate on the remineralization potential of early enamel lesions: An in vitro study

    Directory of Open Access Journals (Sweden)

    Anshul Sharma

    2017-01-01

    Full Text Available Background: Benefits of remineralizing agents in a wide variety of formulations have been proved beneficial in caries management. Casein phosphopeptide-amorphous calcium phosphate (CPP–ACP nanocomplex has been recommended and used as remineralizing agent. Nano-hydroxyapatite (n-HAp is one of the most biocompatible and bioactive material having wide range of application in dentistry, but does it excel better compared to CPP-ACP. Aims: To evaluate and compare the remineralizing efficiency of the paste containing hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate. Settings and Design: The study was an in vitro single blinded study with lottery method of randomization approved by the Institutional Ethics Committee. Materials and methods: 30 non carious premolar teeth. The teeth were demineralized and divided into 2 groups and subjected to remineralization. The samples were analysed for surface hardness and mineral content. Statistical Analysis: Student t’ test and repeated measures of ANOVA was applied. Results: Average hardness in Nano-hydroxyapatite group increased to 340 ± 31.70 SD and 426 ± 50.62 SD for 15 and 30 days respectively and that of (CPP–ACP, 355.83 ± 38.55 SD and 372.67 ± 53.63 SD. The change in the hardness values was not statistically significant with P value of 0.39 (P > 0.05. Calcium and Phosphorous levels increased in both the groups but was not significant. Conclusion: Both the agents used are effective in causing remineralization of enamel. Nano-hydroxyapatite is more effective as compared to Casein phosphopeptide-amorphous calcium phosphate, in increasing the Calcium and Phosphorus content of enamel, and this effect is more evident over a longer treatment period. Key Message: Remineralizing agents are a boon for caries management. With the advent of many formulations it is difficult to clinically select the agent. This study compares the remineralizing potential of Casein

  14. pI-based phosphopeptide enrichment combined with nanoESI-MS.

    Science.gov (United States)

    Hung, Chien-Wen; Kübler, Dieter; Lehmann, Wolf D

    2007-06-01

    IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pI values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to pI is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.

  15. Lyme disease: a selective medium for isolation of the suspected etiological agent, a spirochete.

    Science.gov (United States)

    Johnson, S E; Klein, G C; Schmid, G P; Bowen, G S; Feeley, J C; Schulze, T

    1984-01-01

    A simple procedure with a new selective culture medium for the isolation of the suspected etiological agent of Lyme disease from ticks is described. Live ticks (Ixodes dammini) were ground with a mortar and pestle, and the suspensions were inoculated into a selective and nonselective medium. The selective medium, which contained kanamycin and 5-fluorouracil, yielded positive spirochete cultures from 100% of the pooled ticks and from 79% of the single tick specimens. The isolation rate for the nonselective medium was 0% from the tick pools and 58% from the single tick specimens. PMID:6361065

  16. Selection of Beauveria bassiana and Metarhizium anisopliae Isolates to Control Triatoma infestans

    Directory of Open Access Journals (Sweden)

    Luz Christian

    1998-01-01

    Full Text Available Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%, conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50 varied from 7.1x105 to 4.3x106 conidia/ml, for a B. bassiana isolate (CG 14 and a M. anisopliae isolate (CG 491 respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30°C, compared to 15 and 20°C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity.

  17. Selective microbial genomic DNA isolation using restriction endonucleases.

    Science.gov (United States)

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

  18. A new selective enrichment procedure for isolating Pasteurella multocida from avian and environmental samples

    Science.gov (United States)

    Moore, M.K.; Cicnjak-Chubbs, L.; Gates, R.J.

    1994-01-01

    A selective enrichment procedure, using two new selective media, was developed to isolate Pasteurella multocida from wild birds and environmental samples. These media were developed by testing 15 selective agents with six isolates of P. multocida from wild avian origin and seven other bacteria representing genera frequently found in environmental and avian samples. The resulting media—Pasteurella multocida selective enrichment broth and Pasteurella multocida selective agar—consisted of a blood agar medium at pH 10 containing gentamicin, potassium tellurite, and amphotericin B. Media were tested to determine: 1) selectivity when attempting isolation from pond water and avian carcasses, 2) sensitivity for detection of low numbers of P. multocida from pure and mixed cultures, 3) host range specificity of the media, and 4) performance compared with standard blood agar. With the new selective enrichment procedure, P. multocida was isolated from inoculated (60 organisms/ml) pond water 84% of the time, whereas when standard blood agar was used, the recovery rate was 0%.

  19. An improved culture method for selective isolation of Campylobacter jejuni from wastewater

    Directory of Open Access Journals (Sweden)

    Jinyong Kim

    2016-08-01

    Full Text Available Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently-available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin, to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with Ct value using quantitative real-time PCR (qRT-PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. In particular, rifampicin supplementation and an increased culture temperature (i.e., 42°C had a decisive effect on the selective enrichment of C. jejuni from wastewater. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater.

  20. Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (Araucaria angustifolia).

    Science.gov (United States)

    Ribeiro, Carlos Marcelo; Cardoso, Elke Jurandy Bran Nogueira

    2012-01-20

    Araucaria angustifolia, a unique species of this genus that occurs naturally in Brazil, has a high socio-economic and environmental value and is critically endangered of extinction, since it has been submitted to intense predatory exploitation during the last century. Root-associated bacteria from A. angustifolia were isolated, selected and characterized for their biotechnological potential of growth promotion and biocontrol of plant pathogenic fungi. Ninety-seven strains were isolated and subjected to chemical tests. All isolates presented at least one positive feature, characterizing them as potential PGPR. Eighteen isolates produced indole-3-acetic acid (IAA), 27 were able to solubilize inorganic phosphate, 21 isolates were presumable diazotrophs, with pellicle formation in nitrogen-free culture medium, 83 were phosphatases producers, 37 were positive for siderophores and 45 endospore-forming isolates were antagonistic to Fusarium oxysporum, a pathogen of conifers. We also observed the presence of bacterial strains with multiple beneficial mechanisms of action. Analyzing the fatty acid methyl ester (FAME) and partial sequencing of the 16S rRNA gene of these isolates, it was possible to characterize the most effective isolates as belonging to Bacillaceae (9 isolates), Enterobacteriaceae (11) and Pseudomonadaceae (1). As far as we know, this is the first study to include the species Ewingella americana as a PGPR. Copyright © 2011 Elsevier GmbH. All rights reserved.

  1. Selection of oleuropein-degrading lactic acid bacteria strains isolated from fermenting Moroccan green olives

    Energy Technology Data Exchange (ETDEWEB)

    Ghabbour, N.; Lamzira, Z.; Thonart, P.; Cidalia, P.; Markaouid, M.; Asehraoua, A.

    2011-07-01

    A total of 177 strains of lactic acid bacteria (LAB) were isolated from early-stage Moroccan Picholine green olive fermentation, including Lactobacillus plantarum (44.63%), Lactobacillus pentosus (25.99%), Lactobacillus brevis (9.61%) and Pediococcus pentosaceus (19.77%). All the isolates were screened for their tolerance to olive leaf extract and oleuropein. Most of the isolates (85.3%) were found able to degrade oleuropein, when evaluated by either oleuropein or 5-Bromo-4-chloro-3-indolyl {beta}-D-glucuronide (X-Gluc) as substrates. The biodegradation capacity of the selected strains of each species was confirmed by HPLC analysis. (Author).

  2. Isolation and characterization of Acinetobacter baumannii recovered from Campylobacter selective medium.

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    Dinesh M Fernando

    2016-11-01

    Full Text Available Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is known to cause multidrug resistant infections. This organism has primarily been isolated from clinical environments and its environmental reservoirs remain largely unknown. In the present study, we recovered seven isolates of A. baumannii growing under conditions selective for Campylobacter spp. (microaerophilic at 42 oC and in the presence of antibiotics from dairy cattle manure storage tank or surface water impacted by livestock effluents. Antibiotic susceptibility tests revealed that all of these isolates were less susceptible to at least two different clinically relevant antibiotics, compared to the type strain A. baumannii ATCC17978. Expression of resistance-nodulation-division efflux pumps, an important mechanism of intrinsic resistance in these organisms, was analyzed and adeB was found to be overexpressed in one and adeJ was overexpressed in three isolates. Comparison of these isolates using genomic DNA Macro-Restriction Fragment Pattern Analysis (MRFPA revealed relatively low relatedness among themselves or with some of the clinical isolates from previous studies. This study suggests that A. baumannii isolates are capable of growing under selective conditions for Campylobacter spp. and that this organism can be present in manure and water.

  3. Enzymatic Dissolution of Biocomposite Solids Consisting of Phosphopeptides to Form Supramolecular Hydrogels

    KAUST Repository

    Shi, Junfeng

    2015-10-14

    Enzyme-catalyzed dephosphorylation is essential for biomineralization and bone metabolism. Here we report the exploration of using enzymatic reaction to transform biocomposites of phosphopeptides and calcium (or strontium) ions to supramolecular hydrogels as a mimic of enzymatic dissolution of biominerals. 31P NMR shows that strong affinity between the phosphopeptides and alkaline metal ions (e.g., Ca2+ or Sr2+) induces the formation of biocomposites as precipitates. Electron microscopy reveals that the enzymatic reaction regulates the morphological transition from particles to nanofibers. Rheology confirms the formation of a rigid hydrogel. As the first example of enzyme-instructed dissolution of a solid to form supramolecular nanofibers/hydrogels, this work provides an approach to generate soft materials with desired properties, expands the application of supramolecular hydrogelators, and offers insights to control the demineralization of calcified soft tissues.

  4. The decreased of Streptococcus Mutans growth after topical application of phosphopeptide amorphous calcium phosphate paste

    OpenAIRE

    Tika Faradina Araf; Meirina Gartika; Warta Dewi

    2011-01-01

    Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) paste is a topical application substance that consisted of a series of milk derivative peptide as a result of phosphorylation and has an antibacterial activity. The objective of this research was to find out the difference of Streptococcus mutans growth before and after CPP-ACP paste given topically to child's teeth. The method of the research was a quasi-experiment. Research samples were 10 students of MI Al Falah Islamic Boarding S...

  5. Isolation of cells for selective treatment and analysis using a magnetic microfluidic chip

    KAUST Repository

    Yassine, Omar

    2014-05-01

    This study describes the development and testing of a magnetic microfluidic chip (MMC) for trapping and isolating cells tagged with superparamagnetic beads (SPBs) in a microfluidic environment for selective treatment and analysis. The trapping and isolation are done in two separate steps; first, the trapping of the tagged cells in a main channel is achieved by soft ferromagnetic disks and second, the transportation of the cells into side chambers for isolation is executed by tapered conductive paths made of Gold (Au). Numerical simulations were performed to analyze the magnetic flux and force distributions of the disks and conducting paths, for trapping and transporting SPBs. The MMC was fabricated using standard microfabrication processes. Experiments were performed with E. coli (K12 strand) tagged with 2.8 μm SPBs. The results showed that E. coli can be separated from a sample solution by trapping them at the disk sites, and then isolated into chambers by transporting them along the tapered conducting paths. Once the E. coli was trapped inside the side chambers, two selective treatments were performed. In one chamber, a solution with minimal nutrition content was added and, in another chamber, a solution with essential nutrition was added. The results showed that the growth of bacteria cultured in the second chamber containing nutrient was significantly higher, demonstrating that the E. coli was not affected by the magnetically driven transportation and the feasibility of performing different treatments on selectively isolated cells on a single microfluidic platform.

  6. Genomic signatures of geographic isolation and natural selection in coral reef fishes.

    Science.gov (United States)

    Gaither, Michelle R; Bernal, Moisés A; Coleman, Richard R; Bowen, Brian W; Jones, Shelley A; Simison, W Brian; Rocha, Luiz A

    2015-04-01

    The drivers of speciation remain among the most controversial topics in evolutionary biology. Initially, Darwin emphasized natural selection as a primary mechanism of speciation, but the architects of the modern synthesis largely abandoned that view in favour of divergence by geographic isolation. The balance between selection and isolation is still at the forefront of the evolutionary debate, especially for the world's tropical oceans where biodiversity is high, but isolating barriers are few. Here, we identify the drivers of speciation in Pacific reef fishes of the genus Acanthurus by comparative genome scans of two peripheral populations that split from a large Central-West Pacific lineage at roughly the same time. Mitochondrial sequences indicate that populations in the Hawaiian Archipelago and the Marquesas Islands became isolated approximately 0.5 Ma. The Hawaiian lineage is morphologically indistinguishable from the widespread Pacific form, but the Marquesan form is recognized as a distinct species that occupies an unusual tropical ecosystem characterized by upwelling, turbidity, temperature fluctuations, algal blooms and little coral cover. An analysis of 3737 SNPs reveals a strong signal of selection at the Marquesas, with 59 loci under disruptive selection including an opsin Rh2 locus. While both the Hawaiian and Marquesan populations indicate signals of drift, the former shows a weak signal of selection that is comparable with populations in the Central-West Pacific. This contrast between closely related lineages reveals one population diverging due primarily to geographic isolation and genetic drift, and the other achieving taxonomic species status under the influence of selection. © 2015 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

  7. Selective isolation and differentiation of a stromal population of human embryonic stem cells with osteogenic potential

    DEFF Research Database (Denmark)

    Harkness, Linda M; Mahmood, Amer; Ditzel, Nicholas

    2011-01-01

    between the hESC colonies were isolated by selective adherence to hyaluronic acid-coated plates (100μg/ml) and were characterized using a combination of FACS analysis and staining. The cells were CD44(+), CD29(+), CD73(+), CD166(+), CD146(+), and CD105(+); and, Oct4(-), CD34(-), CD45(-) and CXCR4(-). When...

  8. Fractionation of five technical lignins by selective extraction in green solvents and characterization of isolated fractions

    NARCIS (Netherlands)

    Boeriu, C.G.; Fitigau, F.; Gosselink, R.J.A.; Frissen, A.E.; Stoutjesdijk, J.H.; Peter, F.

    2014-01-01

    Lignins from softwood, hardwood, grass and wheat straw were fractionated by selective extraction at ambient temperature using green solvents like acetone/water solutions of 10, 30, 50, 70 and 90% (v/v) acetone and ethyl acetate. A comparison between the isolated fractions and unfractionated lignins

  9. Genetic polymorphism and natural selection of Duffy binding protein of Plasmodium vivax Myanmar isolates

    Science.gov (United States)

    2012-01-01

    Background Plasmodium vivax Duffy binding protein (PvDBP) plays an essential role in erythrocyte invasion and a potential asexual blood stage vaccine candidate antigen against P. vivax. The polymorphic nature of PvDBP, particularly amino terminal cysteine-rich region (PvDBPII), represents a major impediment to the successful design of a protective vaccine against vivax malaria. In this study, the genetic polymorphism and natural selection at PvDBPII among Myanmar P. vivax isolates were analysed. Methods Fifty-four P. vivax infected blood samples collected from patients in Myanmar were used. The region flanking PvDBPII was amplified by PCR, cloned into Escherichia coli, and sequenced. The polymorphic characters and natural selection of the region were analysed using the DnaSP and MEGA4 programs. Results Thirty-two point mutations (28 non-synonymous and four synonymous mutations) were identified in PvDBPII among the Myanmar P. vivax isolates. Sequence analyses revealed that 12 different PvDBPII haplotypes were identified in Myanmar P. vivax isolates and that the region has evolved under positive natural selection. High selective pressure preferentially acted on regions identified as B- and T-cell epitopes of PvDBPII. Recombination may also be played a role in the resulting genetic diversity of PvDBPII. Conclusions PvDBPII of Myanmar P. vivax isolates displays a high level of genetic polymorphism and is under selective pressure. Myanmar P. vivax isolates share distinct types of PvDBPII alleles that are different from those of other geographical areas. These results will be useful for understanding the nature of the P. vivax population in Myanmar and for development of PvDBPII-based vaccine. PMID:22380592

  10. An Improved Test Selection Optimization Model Based on Fault Ambiguity Group Isolation and Chaotic Discrete PSO

    Directory of Open Access Journals (Sweden)

    Xiaofeng Lv

    2018-01-01

    Full Text Available Sensor data-based test selection optimization is the basis for designing a test work, which ensures that the system is tested under the constraint of the conventional indexes such as fault detection rate (FDR and fault isolation rate (FIR. From the perspective of equipment maintenance support, the ambiguity isolation has a significant effect on the result of test selection. In this paper, an improved test selection optimization model is proposed by considering the ambiguity degree of fault isolation. In the new model, the fault test dependency matrix is adopted to model the correlation between the system fault and the test group. The objective function of the proposed model is minimizing the test cost with the constraint of FDR and FIR. The improved chaotic discrete particle swarm optimization (PSO algorithm is adopted to solve the improved test selection optimization model. The new test selection optimization model is more consistent with real complicated engineering systems. The experimental result verifies the effectiveness of the proposed method.

  11. [Selective-differential nutrient medium "Shewanella IRHLS agar" for isolation of Shewanella genus bacteria].

    Science.gov (United States)

    Sivolodsky, E P

    2015-01-01

    Development of a selective-differential nutrient medium for isolation of Shewanella genus bacteria. 73 strains of Shewanella bacteria (S. algae--3, S. baltica--26, S. putrefaciens--44) and 80 strains of 22 other bacteria genera were used. Shewanella species were identified by methods and criteria proposed by Nozue H. et al., 1992; Khashe S. et al., 1998. Nutrient media "Shewanella IRHLS Agar" for shewanella isolation was developed. Medium selective factors: irgazan DP-300 (I). 0.14-0.2 g/l and rifampicin (R) 0.0005-0.001 g/l. Shevanella colonies were detected by the production of hydrogen sulfide (H), lipase presence (L), lack of sorbitol fermentation (S). The medium suppressed the growth of hydrogen sulfide producers (Salmonella, Proteus) and blocked hydrogen sulfide production by Citrobacter. Growth of Escherichia, Enterobacter, Klebsiella, Shigella, Staphylococcus, Bacillus was also suppressed, Analytical sensitivity of the medium was 1-2 CFU/ml for Shewanella and Stenotrophomonas, Aerombnas, Serratia genera bacteria. 72 strains of Shewanella were isolated from water of Neva river in this medium, 91.7 ± 3.2% of those produced H2S. 1 strain of S. algae was isolated from clinical material. The developed media allows to use it in a complex for Stenotrophomo- nas sp., Aeromonas sp., Serratia sp., Citrobactersp. and Shewanella bacteria isolation.

  12. A 'select and swap' strategy for the isolation of clones with tightly regulated transgenes.

    Science.gov (United States)

    Sullivan, M J; Carpenter, A J; Porter, A C

    2001-03-01

    Increasing numbers of biological problems are being addressed by genetic approaches that rely on inducible expression of transgenes. It is desirable that expression of such a transgene is tightly regulated, from close to zero expression in the 'off' state, to appreciable (at least physiological) expression in the 'on' state. Although there are many examples where tight regulation has been achieved, certain factors, including chromosomal position effects due to random integration of the transgene, often cause suboptimal inducibility and make the isolation of tightly regulated clones difficult and/or laborious. Here we describe a 'select and swap' strategy for the isolation, from a population of stable transfectants, of clones with tightly regulated transgenes. In this approach, a positively and negatively selectable, inducible marker gene is used to select for clones with optimal transgene regulation. After isolation of such clones, the marker gene is swapped with a linked gene of interest by the use of site-specific recombination. To test this strategy we introduced into human cells a plasmid with a tetracycline-inducible bacterial gpt gene linked to a promoterless luciferase gene, isolated clones with tight gpt expression and used the Cre/loxP site-specific recombination system to swap the gpt gene with the luciferase gene. We discuss ways for refining and developing the system and widening its applicability.

  13. Selection on plant male function genes identifies candidates for reproductive isolation of yellow monkeyflowers.

    Directory of Open Access Journals (Sweden)

    Jan E Aagaard

    Full Text Available Understanding the genetic basis of reproductive isolation promises insight into speciation and the origins of biological diversity. While progress has been made in identifying genes underlying barriers to reproduction that function after fertilization (post-zygotic isolation, we know much less about earlier acting pre-zygotic barriers. Of particular interest are barriers involved in mating and fertilization that can evolve extremely rapidly under sexual selection, suggesting they may play a prominent role in the initial stages of reproductive isolation. A significant challenge to the field of speciation genetics is developing new approaches for identification of candidate genes underlying these barriers, particularly among non-traditional model systems. We employ powerful proteomic and genomic strategies to study the genetic basis of conspecific pollen precedence, an important component of pre-zygotic reproductive isolation among yellow monkeyflowers (Mimulus spp. resulting from male pollen competition. We use isotopic labeling in combination with shotgun proteomics to identify more than 2,000 male function (pollen tube proteins within maternal reproductive structures (styles of M. guttatus flowers where pollen competition occurs. We then sequence array-captured pollen tube exomes from a large outcrossing population of M. guttatus, and identify those genes with evidence of selective sweeps or balancing selection consistent with their role in pollen competition. We also test for evidence of positive selection on these genes more broadly across yellow monkeyflowers, because a signal of adaptive divergence is a common feature of genes causing reproductive isolation. Together the molecular evolution studies identify 159 pollen tube proteins that are candidate genes for conspecific pollen precedence. Our work demonstrates how powerful proteomic and genomic tools can be readily adapted to non-traditional model systems, allowing for genome-wide screens

  14. SELECTION OF INDIGENOUS YEAST STRAINS ISOLATED FROM CV. GEWURZTRAMINER FROM ILOK WINEGROWING REGION

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    Dora DUJMOVIĆ

    2008-11-01

    Full Text Available A total of 36 indigenous strains belonging to the Saccharomyces sensu stricto complex isolated from the variety of Gewurztraminer from Ilok winegrowing region were tested with the purpose of making top-quality wines. Microfermentation was applied in order to examine the synthesis of ethyl alcohol and volatile acidity. Killer factor, synthesis of pectinase, ß-glucosidase and H2S was determined in isolated yeast strains. In the selection results, strains RO 1344, RO 1351, RO 1364 were the best evaluated and they should be examined in midifermentation.

  15. The use of titanium dioxide for selective enrichment of phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2......Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic...... chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting...

  16. Selection, characterisation and evaluation of potential probiotic Lactobacillus spp. isolated from poultry droppings.

    Science.gov (United States)

    Asghar, S; Arif, M; Nawaz, M; Muhammad, K; Ali, M A; Ahmad, M D; Iqbal, S; Anjum, A A; Khan, M; Nazir, J

    2016-02-01

    Aim of the present study was to characterise and evaluate probiotic potential of lactobacilli isolated from indigenous poultry. Lactobacilli were isolated from poultry droppings and identified by genus specific polymerase chain reaction and 16S rRNA gene sequencing. Isolates were characterised in vitro by their ability to tolerate low pH and bile salts, phytase activity, antimicrobial activity, antibiotic susceptibility profile, and autoaggregation and coaggregation with poultry gut pathogens. In vivo evaluation of selected isolates was done by their effect on the body weight gain and immune response of broiler chicks. Total of 90, one-day old chicks, were randomly divided in 9 groups and given selected lactobacilli alone and in combinations (108 cfu/bird, daily) from day 7 to day 35. Body weight gain and humoral immune response to New Castle Disease Virus (NDV) vaccine were determined weekly. Three lactobacilli isolates (SMP52, SMP64 and SMP70) were selected as potentially probiotic bacteria on the basis of in vitro characterisation and identified as Lactobacillus crispatus, Lactobacillus casei and L. crispatus, respectively. Chicks supplemented with 'SMP52', 'SMP64', 'SMP70' and 'SMP64+SMP70' and a commercial probiotic product (Protexin) showed significantly higher mean weight gain per bird (1,584±35.2, 1,629±30.6, 1,668±34.7, 1,619±29.5 and 1,576±31.7 g/bird, respectively) as compared to negative control group (1,394±26.7 g/bird), on day 35. SMP 70 also showed significantly higher geometric mean titre against NDV vaccine at day 21 as compared to negative control. It is concluded that L. crispatus SMP52, L. casei SMP64 and L. crispatus SMP70 are potential probiotic candidates which alone or in different combinations may increase body weight of broilers.

  17. Multivariate fault isolation of batch processes via variable selection in partial least squares discriminant analysis.

    Science.gov (United States)

    Yan, Zhengbing; Kuang, Te-Hui; Yao, Yuan

    2017-09-01

    In recent years, multivariate statistical monitoring of batch processes has become a popular research topic, wherein multivariate fault isolation is an important step aiming at the identification of the faulty variables contributing most to the detected process abnormality. Although contribution plots have been commonly used in statistical fault isolation, such methods suffer from the smearing effect between correlated variables. In particular, in batch process monitoring, the high autocorrelations and cross-correlations that exist in variable trajectories make the smearing effect unavoidable. To address such a problem, a variable selection-based fault isolation method is proposed in this research, which transforms the fault isolation problem into a variable selection problem in partial least squares discriminant analysis and solves it by calculating a sparse partial least squares model. As different from the traditional methods, the proposed method emphasizes the relative importance of each process variable. Such information may help process engineers in conducting root-cause diagnosis. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

  18. Selection and characterization of biofuel-producing environmental bacteria isolated from vegetable oil-rich wastes.

    Science.gov (United States)

    Escobar-Niño, Almudena; Luna, Carlos; Luna, Diego; Marcos, Ana T; Cánovas, David; Mellado, Encarnación

    2014-01-01

    Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale.

  19. Selection of Trichoderma isolates for biological control of Sclerotinia minor and S. sclerotiorum in lettuce

    Directory of Open Access Journals (Sweden)

    Luciana Mecatti Elias

    Full Text Available ABSTRACT Lettuce drop is one of the most important and difficult-to-control diseases affecting lettuce in Brazil and worldwide. This study was carried out to select Trichoderma isolates antagonistic to Sclerotinia minor and S. sclerotiorum, aiming to develop biological control for this pathosystem in Brazil. Thirty-one Trichoderma isolates were obtained with the use of baits and were tested under laboratory conditions for their ability to control S. minor and S. sclerotiorum in seedlings of lettuce cultivar Tainá cultured in Petri dishes containing water-agar medium. Subsequently, four isolates effective for control and showing high sporulation under laboratory conditions were evaluated in greenhouse in two experiments carried out with both pathogens in lettuce seedlings of the same cultivar. Twenty-two isolates showed ability to control S. minor and S. sclerotiorum in the in vitro experiments. The isolates tested under greenhouse conditions, identified as T. asperellum (IBLF 897, IBLF 904 and IBLF 914 and T. asperelloides (IBLF 908, reduced lettuce drop of seedlings caused by both pathogens but were more effective against S. minor. Biological control is a promising technology for the management of lettuce drop, especially because S. minor is the predominant species in infested lettuce fields in Brazil.

  20. Isolating E.Coli Bacteriophage from Raw Sewage and Determining its Selectivity to the Host Cell

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    SM Imeni

    2016-05-01

    Full Text Available Introduction: Bacteriophages are viruses that infect and destroy prokaryote cells, specifically the bacteria. They act too selective, so as each bacteriophage affects only on specific type of bacteria. Due to their specific features, bacteriophages can be used as an appropriate substitute for antibiotics in infectious diseases treatment. Therefore, this study aimed to isolate E. coli-specific bacteriophage from raw sewage. Methods: Eight samples of raw sewage, each containing approximately 50 ml of raw sewage with 10 minute gap, were prepared from Zargandeh wastewater treatment plant, Tehran, Iran. The sewages were mixed with Brain-heart infusion medium (BHI as a liquid culture medium in order to let the microorganisms grow. Incubation, purification and determination of bacteria were followed repeatedly to isolate the bacteriophage. Then it was tested on E.coli (ATCC 25922, Enterococcus faecalis (ATCC 19433, Staphylococcus aureus (ATCC 2392, and Yersinia enterocolitica (ATCC 9610 in order to determine the bacteriophage selectivity. Results: The E.coli bacteriophages were successfully isolated from all the eight samples, that were completely able to lyse and destroy E.coli bacterial cells, though no effect was observed on other types of bacteria. Conclusion: The study findings revealed that bacteriophages act selectively. Considering the raise of antibiotic resistance in the world, bacteriophages can serve as a good substitute for antibiotics in treating infectious diseases.

  1. A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast

    Science.gov (United States)

    Noskov, Vladimir N; Kouprina, Natalay; Leem, Sun-Hee; Ouspenski, Ilia; Barrett, J Carl; Larionov, Vladimir

    2003-01-01

    Background Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (~ 60 bp) 5' and 3' gene targeting sequences (hooks). Result TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS) that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3. Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events. Conclusion The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences. PMID:12720573

  2. A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast

    Directory of Open Access Journals (Sweden)

    Barrett J Carl

    2003-04-01

    Full Text Available Abstract Background Transformation-associated recombination (TAR cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (~ 60 bp 5' and 3' gene targeting sequences (hooks. Result TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3. Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events. Conclusion The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.

  3. Comparative evaluation of the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol-containing chewing gum on salivary flow rate, pH and buffering capacity in children: An in vivo study

    OpenAIRE

    Rahul J Hegde; Janhavi B Thakkar

    2017-01-01

    Aim: This study aimed to compare and evaluate the changes in the salivary flow rate, pH, and buffering capacity before and after chewing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol-containing chewing gums in children. Materials and Methods: Sixty children aged between 8 and 12 years were selected for the study. They were randomly divided into Group 1 (CPP-ACP chewing gum) and Group 2 (xylitol-containing chewing gum) comprising thirty children each. Unstimulated and...

  4. DEVELOPMENT AND EVALUATION OF A SELECTIVE AND INDICATIVE MEDIUM FOR ISOLATION OF ACTINOBACILLUS-PLEUROPNEUMONIAE FROM TONSILS

    DEFF Research Database (Denmark)

    Jakobsen, Marianne; Nielsen, Jens

    1995-01-01

    In order to isolate ActinobacillIus pleuropneumoniae from mixed bacterial flora a selective and indicative medium was developed. The optimal concentrations of antibiotics were determined for selective chocolate agar (S-TSA) and selective blood agar (S-MBA) using a set of 25 strains of A. pleuropn......In order to isolate ActinobacillIus pleuropneumoniae from mixed bacterial flora a selective and indicative medium was developed. The optimal concentrations of antibiotics were determined for selective chocolate agar (S-TSA) and selective blood agar (S-MBA) using a set of 25 strains of A...

  5. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    Directory of Open Access Journals (Sweden)

    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  6. Population genetic structure and natural selection of apical membrane antigen-1 in Plasmodium vivax Korean isolates.

    Science.gov (United States)

    Kang, Jung-Mi; Lee, Jinyoung; Cho, Pyo-Yun; Moon, Sung-Ung; Ju, Hye-Lim; Ahn, Seong Kyu; Sohn, Woon-Mok; Lee, Hyeong-Woo; Kim, Tong-Soo; Na, Byoung-Kuk

    2015-11-16

    Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a leading candidate antigen for blood stage malaria vaccine. However, antigenic variation is a major obstacle in the development of an effective vaccine based on this antigen. In this study, the genetic structure and the effect of natural selection of PvAMA-1 among Korean P. vivax isolates were analysed. Blood samples were collected from 66 Korean patients with vivax malaria. The entire PvAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvAMA-1 sequence of each isolate was sequenced and the polymorphic characteristics and effect of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. Thirty haplotypes of PvAMA-1, which were further classified into seven different clusters, were identified in the 66 Korean P. vivax isolates. Domain II was highly conserved among the sequences, but substantial nucleotide diversity was observed in domains I and III. The difference between the rates of non-synonymous and synonymous mutations suggested that the gene has evolved under natural selection. No strong evidence indicating balancing or positive selection on PvAMA-1 was identified. Recombination may also play a role in the resulting genetic diversity of PvAMA-1. This study is the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Korea. Korean PvAMA-1 had limited genetic diversity compared to PvAMA-1 in global isolates. The overall pattern of genetic polymorphism of Korean PvAMA-1 differed from other global isolates and novel amino acid changes were also identified in Korean PvAMA-1. Evidences for natural selection and recombination event were observed, which is likely to play an important role in generating genetic diversity across the PvAMA-1. These results provide useful information for the understanding the population structure of P. vivax circulating in Korea and have important

  7. Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells.

    Science.gov (United States)

    Ozcelebi, F; Miller, L J

    1995-02-17

    The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.

  8. 3-Aminoquinoline/p-coumaric acid as a MALDI matrix for glycopeptides, carbohydrates, and phosphopeptides.

    Science.gov (United States)

    Fukuyama, Yuko; Funakoshi, Natsumi; Takeyama, Kohei; Hioki, Yusaku; Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-Ichirou; Iwamoto, Shinichi; Tanaka, Koichi

    2014-02-18

    Glycosylation and phosphorylation are important post-translational modifications in biological processes and biomarker research. The difficulty in analyzing these modifications is mainly their low abundance and dissociation of labile regions such as sialic acids or phosphate groups. One solution in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is to improve matrices for glycopeptides, carbohydrates, and phosphopeptides by increasing the sensitivity and suppressing dissociation of the labile regions. Recently, a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) (3-AQ/CHCA), introduced by Kolli et al. in 1996, has been reported to increase sensitivity for carbohydrates or phosphopeptides, but it has not been systematically evaluated for glycopeptides. In addition, 3-AQ/CHCA enhances the dissociation of labile regions. In contrast, a liquid matrix 1,1,3,3-tetramethylguanidium (TMG, G) salt of p-coumaric acid (CA) (G3CA) was reported to suppress dissociation of sulfate groups or sialic acids of carbohydrates. Here we introduce a liquid matrix 3-AQ/CA for glycopeptides, carbohydrates, and phosphopeptides. All of the analytes were detected as [M + H](+) or [M - H](-) with higher or comparable sensitivity using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-dihydroxybenzoic acid (2,5-DHB). The sensitivity was increased 1- to 1000-fold using 3-AQ/CA. The dissociation of labile regions such as sialic acids or phosphate groups and the fragmentation of neutral carbohydrates were suppressed more using 3-AQ/CA than using 3-AQ/CHCA or 2,5-DHB. 3-AQ/CA was thus determined to be an effective MALDI matrix for high sensitivity and the suppression of dissociation of labile regions in glycosylation and phosphorylation analyses.

  9. Iron oxide nanoparticle coating of organic polymer-based monolithic columns for phosphopeptide enrichment

    Czech Academy of Sciences Publication Activity Database

    Křenková, Jana; Foret, František

    2011-01-01

    Roč. 34, 16-17 (2011), s. 2106-2112 ISSN 1615-9306 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GPP206/11/P004 Grant - others:Jihomoravský kraj(CZ) 2SGA2721 Program:2SGA Institutional research plan: CEZ:AV0Z40310501 Keywords : iron oxide nanoparticles * monolithic column * phosphopeptide enrichment Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.733, year: 2011

  10. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

    DEFF Research Database (Denmark)

    Molina, Henrik; Horn, David M; Tang, Ning

    2007-01-01

    Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total...... of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 ( approximately 80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more...

  11. Structural insights into the recognition of phosphopeptide by the FHA domain of kanadaptin

    DEFF Research Database (Denmark)

    Xu, Qingping; Deller, Marc C; Nielsen, Tine K

    2014-01-01

    Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed...... with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins...

  12. Compost as a source of microbial isolates for the bioremediation of heavy metals: In vitro selection

    Energy Technology Data Exchange (ETDEWEB)

    Vargas-Garcia, Maria del Carmen, E-mail: mcvargas@ual.es; Lopez, Maria Jose, E-mail: mllopez@ual.es; Suarez-Estrella, Francisca, E-mail: fsuarez@ual.es; Moreno, Joaquin, E-mail: jcasco@ual.es

    2012-08-01

    Heavy metal pollution has become a major environmental concern nowadays and the bioremediation of polluted habitats is an increasingly popular strategy due to both its efficiency and safety. A screening and selection protocol based on different composting processes was designed in order to isolate heavy metal-resistant microorganisms. A collection of 51 microorganisms was obtained and most of them showed the capability to tolerate heavy metals in multi-polluted aqueous systems (Cd(II), Cr(VI), Ni, Pb, Zn(II)), as well as to remove them. The highest detoxification ratios were observed for Pb. Some of the isolates detoxifying more than a 90% of this metal, while the other metals were removed in a range between 20% and 60%. The best isolates (Graphium putredinis, Fusarium solani, Fusarium sp. and Penicillium chrysogenum) were further assayed in order to determine the predominant removal mechanism and the potential use of their dead biomass as a biosorbent. Intracellular accumulation was the prevalent mechanism for most isolates and metals, with the exception of Ni. In this case, the proportion removed by extracellular adsorption was similar or even higher than that removed by intracellular accumulation. Thus, the efficiency of living cells was higher than that of dead biomass except in the case of Ni. - Highlights: Black-Right-Pointing-Pointer Composting is a good reservoir for the isolation of HM-resistant microorganisms. Black-Right-Pointing-Pointer Pb was the most removed heavy metal in multi-polluted aqueous systems. Black-Right-Pointing-Pointer Intracellular accumulation was the predominant mechanism for heavy metal removal. Black-Right-Pointing-Pointer Graphium putredinis, which detoxifies organic pollutants, was the most efficient isolate.

  13. Efficacy of Syringe Filtration for the Selective Isolation of Campylobacter from Chicken Carcass Rinse.

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Hong-Seok; Kim, Dong-Hyeon; Kim, Young-Ji; Sung, Kidon; Kim, Hyunsook; Seo, Kun-Ho

    2017-06-01

    We investigated the efficacy of syringe filtration for selective isolation of Campylobacter from chicken carcass rinse by combining syringe filtration with the conventional culture method. Whole chicken carcass rinses were incubated in Bolton enrichment broth, set aside or subjected to syringe filtration, and streaked on Campy-Cefex agar with or without cefoperazone antibiotic supplement. Compared with the conventional method without filtration, 0.65-μm-pore-size syringe filtration resulted in a significantly higher number of Campylobacter-positive samples (23.8 to 37.5% versus 70.0 to 72.5%; P Campylobacter (93.8% versus 6.3 to 26.3%), and a lower growth index (1 = growth of a few colonies; 2 = growth of colonies on about half of the plate; and 3 = growth on most of the plate) for competing microbiota (2.9 to 3.0 versus 1.2 to 1.4). When syringe filtration was applied, agar plates containing the antibiotic had significantly less contamination (6.3% versus 26.3%; P Campylobacter isolation rate was similar (P > 0.05). Syringe filtration combined with conventional enrichment improved the rate and selectivity of Campylobacter isolation from chicken carcasses.

  14. Isolation and Fatty Acid Profile of Selected Microalgae Strains from the Red Sea for Biofuel Production

    Directory of Open Access Journals (Sweden)

    Khalid M. Abu-Salah

    2013-05-01

    Full Text Available The isolation of lipid-rich autochthonous strains of microalgae is a crucial stage for the development of a microalgae-based biofuel production plant, as these microalgae already have the necessary adaptations to withstand competition, predation and the temperatures observed at each production site. This is particularly important in extreme climates such as in Saudi Arabia. Resorting to fluorescence activated cell sorting (FACS we screened for and isolated several microalgal strains from samples collected from the Red Sea. Relying on the fluorescence of BODIPY 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diazasindacene and growth performance, four promising candidates were identified and the total lipid content and fatty acid profile was assessed for biofuels production. Selected isolates were classified as chlorophytes, belonging to three different genera: Picochlorum, Nannochloris and Desmochloris. The lipid contents were assessed microscopically by means of BODIPY 505/515-associated fluorescence to detect intracellular lipid bodies, which revealed several lipid drops in all selected strains. This result was confirmed by lipid gravimetric determination, which demonstrated that all strains under study presented inner cell lipid contents ranging from 20% to 25% of the biomass dry weight. Furthermore, the fatty acid methyl esters profile of all strains seems ideal for biodiesel production due to a low degree of polyunsaturated fatty acid methyl esters and high amount of palmitic and oleic acids.

  15. Virulence potential of Enterococcus gallinarum strains isolated from selected Nigerian traditional fermented foods

    Directory of Open Access Journals (Sweden)

    IYABO C. OLADIPO

    2014-08-01

    Full Text Available Five Enterococcus isolates from some Nigerian traditional fermented foods were identified as Enterococcus gallinarum by using phenotypic and genotypic tests. Safety properties such as antibiotic susceptibility, virulence gene detection, haemolysin, gelatinase and bacteriocin production were determined using standard methods. There was no resistance to clinically relevant antibiotics. Virulence gene for collagen binding antigen and aggregation substance were detected in 60% of the E. gallinarum strains; while surface adhesin was detected in 20%, but none of the strains had cytolysin activator and gelatinase. Phenotype characterizations of the E. gallinarum isolates indicated that none of the isolates produced haemolysin and gelatinase. Enterococcus gallinarum C103 and U82 had no antimicrobial activity against all the selected bacteria pathogens while E. gallinarum W184, T71 and W21 were active against some of the indicator bacteria pathogens. Only E. gallinarum T71 and W21 showed broad spectra of antimicrobial activity. Combination of virulence factors did not appear in these food isolates. Therefore, these strains particularly the two strains with high spectra of antimicrobial activity could be exploited as functional starters in foods.

  16. Population genetic structure and natural selection of Plasmodium falciparum apical membrane antigen-1 in Myanmar isolates.

    Science.gov (United States)

    Kang, Jung-Mi; Lee, Jinyoung; Moe, Mya; Jun, Hojong; Lê, Hương Giang; Kim, Tae Im; Thái, Thị Lam; Sohn, Woon-Mok; Myint, Moe Kyaw; Lin, Khin; Shin, Ho-Joon; Kim, Tong-Soo; Na, Byoung-Kuk

    2018-02-07

    Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is one of leading blood stage malaria vaccine candidates. However, genetic variation and antigenic diversity identified in global PfAMA-1 are major hurdles in the development of an effective vaccine based on this antigen. In this study, genetic structure and the effect of natural selection of PfAMA-1 among Myanmar P. falciparum isolates were analysed. Blood samples were collected from 58 Myanmar patients with falciparum malaria. Full-length PfAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. PfAMA-1 sequence of each isolate was sequenced. Polymorphic characteristics and effect of natural selection were analysed with using DNASTAR, MEGA4, and DnaSP programs. Polymorphic nature and natural selection in 459 global PfAMA-1 were also analysed. Thirty-seven different haplotypes of PfAMA-1 were identified in 58 Myanmar P. falciparum isolates. Most amino acid changes identified in Myanmar PfAMA-1 were found in domains I and III. Overall patterns of amino acid changes in Myanmar PfAMA-1 were similar to those in global PfAMA-1. However, frequencies of amino acid changes differed by country. Novel amino acid changes in Myanmar PfAMA-1 were also identified. Evidences for natural selection and recombination event were observed in global PfAMA-1. Among 51 commonly identified amino acid changes in global PfAMA-1 sequences, 43 were found in predicted RBC-binding sites, B-cell epitopes, or IUR regions. Myanmar PfAMA-1 showed similar patterns of nucleotide diversity and amino acid polymorphisms compared to those of global PfAMA-1. Balancing natural selection and intragenic recombination across PfAMA-1 are likely to play major roles in generating genetic diversity in global PfAMA-1. Most common amino acid changes in global PfAMA-1 were located in predicted B-cell epitopes where high levels of nucleotide diversity and balancing natural selection were found. These results highlight the

  17. Selection of tomato plants resistant to a local Polish isolate of tomato spotted wilt virus (TSWV).

    Science.gov (United States)

    Czech, Andrzej S; Szklarczyk, Marek; Gajewski, Zbigniew; Zukowska, Ewa; Michalik, Barbara; Kobyłko, Tadeusz; Strzałka, Kazimierz

    2003-01-01

    We found that the Sw-5 gene confers resistance to one of the Polish isolates of tomato spotted wilt virus (TSWV). A series of tomato breeding accessions was analysed along with standards of resistance and susceptibility to TSWV. The presence of the Sw-5 gene was determined using the available PCR marker. Subsequently plants from these accessions were grown in the presence of the TSWV isolate from Poland. Some of them developed severe symptoms of the TSWV disease. Expression of the virus proteins was also assayed in tissues of the investigated plants. We found general agreement between either lack or presence of the disease symptoms, virus proteins and resistance gene. Some observed discrepancies of these data are also discussed. Our results indicate that marker-assisted selection can be used for breeding of the TSWV-resistant tomato in Poland.

  18. Adding casein phosphopeptide-amorphous calcium phosphate to sports drinks to eliminate in vitro erosion.

    Science.gov (United States)

    Ramalingam, L; Messer, L B; Reynolds, E C

    2005-01-01

    Enamel erosion can occur with frequent consumption of sports drinks. The purpose of this study was to determine a minimal concentration of casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP) which when added to a sports drink would eliminate such erosion in vitro. Human enamel specimens were immersed in: (1) the sports drink Powerade; (2) Poweradeplus 4 concentrations of CPP-ACP (0.063%, 0.09%, 0.125%, 0.25%); or (3) double deionized water. Windows of test and control enamel were profiled, and the enamel surface characteristics were examined under scanning electron microscopy (SEM). The pH of test solutions increased and the titratable acidity decreased with increasing CPP-ACP concentrations. Erosive step lesions occurred in specimens immersed in Powerade (mean depth=38.70kA +/- 5.60), which were eliminated by the addition of CPP-ACP to Powerade at all test concentrations except 0.063% CPP-ACP. Microscopic surface irregularities on test enamel were observed, apparent as adherent granules or globules. These may represent redeposited mineral phases following mobilization of calcium and phosphate from CPP-ACP. Tasters in a taste panel could not distinguish Powerade from Powerade plus 0.125% CPP-ACP. Adding casein phosphopeptide-stabilized amorphous calcium phosphate to the sports drink Powerade significantly reduced the beverage's erosivity without affecting the product's taste.

  19. A lab-on-a-chip for monolith-based preconcentration and electrophoresis separation of phosphopeptides.

    Science.gov (United States)

    Araya-Farias, Monica; Dziomba, Szymon; Carbonnier, Benjamin; Guerrouache, Mohamed; Ayed, Ichraf; Aboud, Nacera; Taverna, Myriam; Tran, N Thuy

    2017-01-26

    A microdevice combining online preconcentration and separation of phosphopeptides was developed in a glass microchip. An ethylene glycol methacrylate phosphate (EGMP), acrylamide (AM) and bisacrylamide (BAA) based monolith was synthesized within microchannels through a photo-driven process. Morphological investigations revealed a homogeneous monolithic structure composed of uniform nodules (∼0.8 μm), with a large pore volume (0.62 cm3 g-1) and sufficiently high specific surface area (34.1 m2 g-1). These features make the monolith particularly interesting for preconcentration purposes. Immobilization of Zr4+ ions on the phosphate groups present at the poly(EGMP-co-AM-co-BAA) monolith surface leads to immobilized metal affinity chromatography support. This monolith-Zr4+ showed a great capacity to capture phosphopeptides. Successful preconcentration and separation of a mixture of ERK2 derived peptides differing only by their phosphorylation degree and sites could be achieved with signal enhancement factors between 340 and 910 after only 7 min of preconcentration. This integrated microdevice represents a novel approach for phosphoproteomic applications.

  20. Remineralization of early enamel lesions using casein phosphopeptide amorphous calcium Phosphate: An ex-vivo study

    Directory of Open Access Journals (Sweden)

    Ruchi Vashisht

    2010-01-01

    Full Text Available Objective: This study aimed at qualitatively evaluating the remineralization potential of casein phosphopeptide amorphous calcium phosphate on artificial early enamel lesions in an ex-vivo scenario by observing the treated tooth surface using a scanning electron microscope (SEM. Materials and Methods: This randomized study was conducted on 10 subjects undergoing orthodontic treatment with premolar extraction as part of their treatment. Artificial white lesions were created with the application of 37% phosphoric acid for 20 mins. Teeth were then divided into two groups: one experimental and the other control. Customised orthodontic band with a window was luted with intermediate restorative material in the experimental group whereas in the control group, band without a window was luted. The casein phosphopeptide amorphous calcium phosphate (GC TOOTH MOUSSE paste was then applied on the window region of the experimental group for 3 mins thrice daily after meals for 14 days, whereas no paste was applied in the control group. After 14 days, teeth were extracted and viewed under an SEM. Results: The study groups showed remineralization of the lesions as compared with the control group in most of the samples. Conclusion: Casein phophopeptide could significantly remineralize the artificial enamel lesions in vivo.

  1. Improved detection of phosphopeptides by negative ion matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge co-matrix

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Shu [State Key Laboratory for Chirosciences and Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Kowloon (Hong Kong); Yao Zhongping, E-mail: bczpyao@inet.polyu.edu.hk [State Key Laboratory for Chirosciences and Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Kowloon (Hong Kong)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Use of ATT/DMAN/DHC matrix for detection of phosphopeptides by negative MALDI-MS. Black-Right-Pointing-Pointer Lower limit of detection. Black-Right-Pointing-Pointer Reduced signal suppression effects. Black-Right-Pointing-Pointer Improved position-to-position reproducibility. - Abstract: Analysis of phosphopeptides is an important task in proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a technique very commonly used for such a purpose. Analysis of phosphopeptides by MALDI-MS is, however, still a challenging task due to the low ionization efficiency of phosphopeptides. In this study, we reported that by using a proton sponge 1,8-bis(dimethyl-amino)naphthalene (DMAN) as a co-matrix, detection of phosphopeptides by negative ion MALDI-MS could be greatly improved. Combination of DMAN with another matrix 6-aza-2-thiothymine (ATT) and additive diammonium hydrogen citrate (DHC) allowed much lower limit of detection, significantly reduced signal suppression effects and improved position-to-position reproducibility for detection of phosphopeptides by negative ion MALDI-MS. Potential applications of the matrix system in qualification of phosphopeptides and analysis of proteolytic digests of phosphorylated proteins were also demonstrated in this study.

  2. Selective isolation of large chromosomal regions by transformation-associated recombination cloning for structural and functional analysis of mammalian genomes.

    Science.gov (United States)

    Kouprina, Natalay; Noskov, Vladimir N; Larionov, Vladimir

    2006-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-size genes and genomic loci as circular yeast artificial chromosomes in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. This chapter describes a basic protocol of gene isolation by TAR, as well as a method of conversion of TAR isolates into bacterial artificial chromosomes.

  3. Electro-membrane filtration for the selective isolation of bioactive peptides from an ?s2-casein hydrolysate

    NARCIS (Netherlands)

    Bargeman, Gerrald; Houwing, Joukje; Recio, Isidra; Koops, G.H.; van der Horst, Caroline

    2002-01-01

    For the isolation of the ingredients required for functional foods and nutraceuticals generally membrane filtration has too low a selectivity and chromatography is (too) expensive. Electro-membrane filtration (EMF) seems to be a breakthrough technology for the isolation of charged nutraceutical

  4. SENSITIVITY OF MOLDS ISOLATED FROM WAREHOUSES OF FOOD PRODUCTION FACILITY ON SELECTED ESSENTIAL OILS

    Directory of Open Access Journals (Sweden)

    Łukasz Kręcidło

    2015-07-01

    Full Text Available Storage of raw materials is one of steps in food production chain. The aim of this study was to estimate the influence of selected essential oils on the growth of four fungal strains: Trichoderma viride, Rhizomucor miehei, Penicillium chrysogenum, Penicillium janthinellum. Strains were isolated from warehouses of the food production facility. Selected essential oils: thyme oil, rosewood oil and rosemary oil were used to assess antifungal activity. Chemical composition of essential oils was determined by Gas Chromatography-Mass Spectroscopy (GC-MS. Antifungal activity of essential oils was estimated in relative to peracetic acid (PAA and sterile water with Tween 80 (0,5%. The influence of essential oils on fungal growth was carried by medium poisoning method. Increment of fungal mycelium was measured every day by 10 days. The thyme essential oils totally inhibited fungal growth in the lowest concentration of 1 mm3·cm-3. The most resistant strain was Penicillium janthinellum.

  5. Cluster Head Selection in a Homogeneous Wireless Sensor Network Ensuring Full Connectivity with Minimum Isolated Nodes

    Directory of Open Access Journals (Sweden)

    Tapan Kumar Jain

    2014-01-01

    Full Text Available The research work proposes a cluster head selection algorithm for a wireless sensor network. A node can be a cluster head if it is connected to at least one unique neighbor node where the unique neighbor is the one that is not connected to any other node. If there is no connected unique node then the CH is selected on the basis of residual energy and the number of neighbor nodes. With the increase in number of clusters, the processing energy of the network increases; hence, this algorithm proposes minimum number of clusters which further leads to increased network lifetime. The major novel contribution of the proposed work is an algorithm that ensures a completely connected network with minimum number of isolated nodes. An isolated node will remain only if it is not within the transmission range of any other node. With the maximum connectivity, the coverage of the network is automatically maximized. The superiority of the proposed design is verified by simulation results done in MATLAB, where it clearly depicts that the total numbers of rounds before the network dies out are maximum compared to other existing protocols.

  6. Semi-selective culture medium for Exserohilum turcicum isolation from corn seeds

    Directory of Open Access Journals (Sweden)

    Roberto Luis De Rossi

    2014-06-01

    Full Text Available Northern corn leaf blight, caused by Exserohilum turcicum (Et, is a disease of widespread occurrence in regions where corn, sweetcorn and popcorn are grown. This disease has great potential to cause damage and has been studied for years, but the association of its causal agent with seeds remains unconfirmed. Thus, the availability of a sensitive method to detect and quantify the inoculum in seeds, even at low incidence, is essential. The aim of this study was to develop a method to detect and quantify the presence of the fungus infecting and infesting corn and popcorn seeds. Artificially and naturally infected seeds were employed to develop the medium. The semi-selective medium was composed of carbendazim (active ingredient (60 mg/L, captan (30 mg/L, streptomycin sulfate (500 mg/L and neomycin sulfate (600 mg/L aggregated to the medium lactose casein hydrolysate agar medium. By using this, Et was detected in naturally infected corn seeds, showing 0.124% incidence, in four out of ten analyzed samples. In addition, 1.04 conidia were detected per infested seed. By means of isolation, pathogenicity test, morphological characterization and comparison with descriptions of the species in the literature, the fungus isolated from the seeds was confirmed to be Et. Both infection and infestation were considered low; thus, for studies of Et detection in corn seeds, the use of semi-selective medium and more than 1,200 seeds/sample is suggested.

  7. Isolation, Characterization, and Selection of Molds Associated to Fermented Black Table Olives

    Directory of Open Access Journals (Sweden)

    Simona L. Bavaro

    2017-07-01

    Full Text Available Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly identified as Penicillium crustosum (21, P. roqueforti (29, P. paneum (1, P. expansum (6, P. polonicum (2, P. commune (1. A group of 20 selected isolates was subjected to technological (beta-glucosidase, cellulolytic, ligninolytic, pectolytic, and xylanolytic activities; proteolytic enzymes and safety (biogenic amines and secondary metabolites, including mycotoxins characterization. Combining both technological (presence of desired and absence of undesired enzymatic activities and safety aspects (no or low production of biogenic amines and regulated mycotoxins, it was possible to select six strains with biotechnological interest. These are putative candidates for future studies as autochthonous co-starters with yeasts and lactic acid bacteria for black table olive production.

  8. Semi-selective media for the isolation of Phaeomoniella chlamydospora from soil and vine wood

    Directory of Open Access Journals (Sweden)

    M.L. Tello

    2009-05-01

    Full Text Available Two semi-selective culture media, F10S (PDA + folpet 10 ppm + streptomycin sulphate 1 g l-1 and RB150S (PDA + rose bengal 150 ppm + streptomycin sulphate 1 g l-1, were developed for the isolation of the phytopathogenic fungus Phaeomoniella chlamydospora from soil samples and vine tissues. The media were selected so that they would allow proper growth of the pathogen and would partially inhibit eleven other common fungal genera. Eight antifungal agents were tested: Folpan (a.i. folpet, Captazel (a.i. captan, Benlate (a.i. benomyl, Chipco (a.i. iprodione, Switch (a.i. cyprodinil + fl udioxonil, rose bengal, and the bactericidal antibiotic streptomycin sulphate at several doses. Recovery of Pa. chlamydospora from wood samples was 40% better on RB150S and 50% better on F10S than on PDA, while the contaminants were reduced by 42% with RB150S, and by 48% with F10S. Pathogen reisolation from artificially contaminated soil samples was improved with F10S, while RB150S facilitated pathogen detection in samples containing moderate amounts of Rhizopus, Trichoderma, Penicillium, Alternaria or Trichoderma or in soils heavily contaminated with bacteria. F10S and RB150S improved the isolation of Pa. chlamydospora from wood and soil and can be used as alternatives to current culture media.

  9. An in vitro comparison of casein phosphopeptide-amorphous calcium phosphate paste, casein phosphopeptide-amorphous calcium phosphate paste with fluoride and casein phosphopeptide-amorphous calcium phosphate varnish on the inhibition of demineralization and promotion of remineralization of enamel

    Directory of Open Access Journals (Sweden)

    Prachi Jayesh Thakkar

    2017-01-01

    Full Text Available Aim: This study aims to determine and compare the extent of inhibition of demineralization and promotion of remineralization of permanent molar enamel with and without application of three remineralizing agents. Materials and Methods: Forty extracted permanent molars were randomly divided into two groups 1 and 2, longitudinally sectioned into four and divided into subgroups A, B, C, and D. The sections were coated with nail varnish leaving a window of 3 mm × 3 mm. All sections of Group 1 were treated with their respective subgroup-specific agent: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP paste for subgroup A, CPP-amorphous calcium phosphate fluoride (ACPF paste for subgroup B, CPP-ACPF varnish for subgroup C and subgroup D served as a control. The sections were then subjected to demineralization for 12 days following which lesional depth was measured under the stereomicroscope. All the sections of Group 2 were subjected to demineralization for 12 days, examined for lesional depth, then treated with their respective subgroup specific agents and immersed in artificial saliva for 7 days. The sections were then examined again under the stereomicroscope to measure the lesional depth. Results: CPP-ACPF varnish caused significant inhibition of demineralization. All three agents showed significant remineralization of previously demineralized lesions. However, CPP-ACPF varnish showed the greatest remineralization, followed by CPP-ACPF paste and then CPP-ACP paste. Conclusion: This study shows that CPP-ACPF varnish is effective in preventing demineralization as well as promoting remineralization of enamel. Thus, it can be used as an effective preventive measure for pediatric patients where compliance with the use of tooth mousse may be questionable.

  10. An in vitro comparison of casein phosphopeptide-amorphous calcium phosphate paste, casein phosphopeptide-amorphous calcium phosphate paste with fluoride and casein phosphopeptide-amorphous calcium phosphate varnish on the inhibition of demineralization and promotion of remineralization of enamel.

    Science.gov (United States)

    Thakkar, Prachi Jayesh; Badakar, Chandrashekhar M; Hugar, Shivayogi M; Hallikerimath, Seema; Patel, Punit M; Shah, Parin

    2017-01-01

    This study aims to determine and compare the extent of inhibition of demineralization and promotion of remineralization of permanent molar enamel with and without application of three remineralizing agents. Forty extracted permanent molars were randomly divided into two groups 1 and 2, longitudinally sectioned into four and divided into subgroups A, B, C, and D. The sections were coated with nail varnish leaving a window of 3 mm × 3 mm. All sections of Group 1 were treated with their respective subgroup-specific agent: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste for subgroup A, CPP-amorphous calcium phosphate fluoride (ACPF) paste for subgroup B, CPP-ACPF varnish for subgroup C and subgroup D served as a control. The sections were then subjected to demineralization for 12 days following which lesional depth was measured under the stereomicroscope. All the sections of Group 2 were subjected to demineralization for 12 days, examined for lesional depth, then treated with their respective subgroup specific agents and immersed in artificial saliva for 7 days. The sections were then examined again under the stereomicroscope to measure the lesional depth. CPP-ACPF varnish caused significant inhibition of demineralization. All three agents showed significant remineralization of previously demineralized lesions. However, CPP-ACPF varnish showed the greatest remineralization, followed by CPP-ACPF paste and then CPP-ACP paste. This study shows that CPP-ACPF varnish is effective in preventing demineralization as well as promoting remineralization of enamel. Thus, it can be used as an effective preventive measure for pediatric patients where compliance with the use of tooth mousse may be questionable.

  11. The COX-2 Selective Blocker Etodolac Inhibits TNFα-Induced Apoptosis in Isolated Rabbit Articular Chondrocytes

    Directory of Open Access Journals (Sweden)

    Hiroshi Matsuura

    2013-09-01

    Full Text Available Chondrocyte apoptosis contributes to the disruption of cartilage integrity in osteoarthritis (OA. Recently, we reported that activation of volume-sensitive Cl− current (ICl,vol mediates cell shrinkage, triggering apoptosis in rabbit articular chondrocytes. A cyclooxygenase (COX blocker is frequently used for the treatment of OA. In the present study, we examined in vitro effects of selective blockers of COX on the TNFα-induced activation of ICl,vol in rabbit chondrocytes using the patch-clamp technique. Exposure of isolated chondrocytes to TNFα resulted in an obvious increase in membrane Cl− conductance. The TNFα-evoked Cl− current exhibited electrophysiological and pharmacological properties similar to those of ICl,vol. Pretreatment of cells with selective COX-2 blocker etodolac markedly inhibited ICl,vol activation by TNFα as well as subsequent apoptotic events such as apoptotic cell volume decrease (AVD and elevation of caspase-3/7 activity. In contrast, a COX-1 blocker had no effect on the decrease in cell volume or the increase in caspase-3/7 activity induced by TNFα. Thus, the COX-2-selective blocker had an inhibitory effect on TNFα-induced apoptotic events, which suggests that this drug would have efficacy for the treatment of OA.

  12. Comparative antimicrobial efficacy of selected root canal irrigants on commonly isolated microorganisms in endodontic infection.

    Science.gov (United States)

    Dubey, Sandeep; Saha, Suparna Ganguly; Rajkumar, Balakrishnan; Dhole, Tapan Kumar

    2017-01-01

    This study aims to evaluate and compare the antimicrobial efficacy of three selected root canal irrigants (BioPure MTAD, metronidazole, aztreonam) against microbes commonly isolated from polymicrobial microbiota of root canal infection. This study was designed with four experimental groups (Group I - Bacteroides fragilis, Group II - Propionibacterium acnes, Group III - Enterococcus faecalis, Group IV - Candida albicans) based on the microbes selected for the study. Group I and Group II bacteria were used to compare and evaluate antimicrobial effect of BioPure MTAD, metronidazole, aztreonam, and normal saline. Group III and Group IV bacteria were used to compare and evaluate antimicrobial efficacy of BioPure MTAD, aztreonam, and normal saline. Normal saline was used as a control irrigant in this study. Agar disc diffusion method was applied to assess and compare the antimicrobial action of selected irrigants. Metronidazole was found to be the most effective root canal irrigant against B. fragilis and P. acnes among the tested irrigants. Mean zone of inhibition against E. faecalis has been shown to be maximum by BioPure MTAD, followed by aztreonam. Antifungal effect against C. albicans was only shown by BioPure MTAD. Overall, BioPure MTAD is the most effective root canal irrigant as it has shown an antibacterial effect against all the tested microorganisms. However, metronidazole showed maximum antibacterial effect against obligate anaerobes. Aztreonam also showed an antibacterial effect in the present study, raising its possibility to be used as a root canal irrigant in the future.

  13. Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Graham, Mark E; Robinson, Phillip J

    2004-01-01

    , or peptides altered in hydrophilicity such as phosphopeptides. We used microcolumns to compare the ability of RP resin or graphite powder to retain phosphopeptides. A number of standard phosphopeptides and a biologically relevant phosphoprotein, dynamin I, were analyzed. MS revealed that some phosphopeptides...... did not bind the RP resin but were retained efficiently on the graphite. Those that did bind the RP resin often produced much stronger signals from the graphite powder. In particular, the method revealed a doubly phosphorylated peptide in a tryptic digest of dynamin I purified from rat brain nerve...... terminals. The detection of this peptide was greatly enhanced by graphite micropurification. Sequencing by tandem MS confirmed the presence of phosphate at both Ser-774 and Ser-778, while a singly phosphorylated peptide was predominantly phosphorylated only on Ser-774. The method further revealed a singly...

  14. Spatially heterogeneous environmental selection strengthens evolution of reproductively isolated populations in a Dobzhansky-Muller system of hybrid incompatability.

    Directory of Open Access Journals (Sweden)

    Samuel A Cushman

    2016-11-01

    Full Text Available Within-species hybrid incompatibility can arise when combinations of alleles at more than one locus have low fitness but where possession of one of those alleles has little or no fitness consequence for the carriers. Limited dispersal with small numbers of mate potentials alone can lead to the evolution of clusters of reproductively isolated genotypes despite the absence of any geographical barriers or heterogeneous selection. In this paper, we explore how adding heterogeneous natural selection on the genotypes (e.g., gene environment associations that are involved in reproductive incompatibility affects the frequency, size and duration of evolution of reproductively isolated clusters. We conducted a simulation experiment that varied landscape heterogeneity, dispersal ability and strength of selection in a continuously distributed population. In our simulations involving spatially heterogeneous selection, strong patterns of adjacency of mutually incompatible genotypes emerged such that these clusters were truly reproductively isolated from each other, with no reproductively compatible bridge individuals in the intervening landscape to allow gene flow between the clusters. This pattern was strong across levels of gene flow and strength of selection, suggesting that even relatively weak selection acting in the context of strong gene flow may produce reproductively isolated clusters that are large and persistent, enabling incipient speciation in a continuous population without geographic isolation.

  15. Isolation and in vitro selection of actinomycetes strains as potential probiotics for aquaculture

    National Research Council Canada - National Science Library

    Bernal, Milagro García; Campa-Córdova, Ángel Isidro; Saucedo, Pedro Enrique; González, Marlen Casanova; Marrero, Ricardo Medina; Mazón-Suástegui, José Manuel

    2015-01-01

    .... Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis...

  16. Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

    Science.gov (United States)

    Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

    2014-12-05

    As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

  17. Continuous labeling of circulating tumor cells with microbeads using a vortex micromixer for highly selective isolation.

    Science.gov (United States)

    Lin, Ming Xian; Hyun, Kyung-A; Moon, Hui-Sung; Sim, Tae Seok; Lee, Jeong-Gun; Park, Jae Chan; Lee, Soo Suk; Jung, Hyo-Il

    2013-02-15

    Circulating tumor cells (CTCs) are identified in transit within the blood stream of cancer patients and have been proven to be a main cause of metastatic disease. Current approaches for the size-based isolation of CTCs have encountered technical challenges as some of the CTCs have a size similar to that of leukocytes and therefore CTCs are often lost in the process. Here, we propose a novel strategy where most of the CTCs are coated by a large number of microbeads to amplify their size to enable complete discrimination from leukocytes. In addition, all of the microbead labeling processes are carried out in a continuous manner to prevent any loss of CTCs during the isolation process. Thus, a microfluidic mixer was employed to facilitate the efficient and selective labeling of CTCs from peripheral blood samples. By generating secondary vortex flows called Taylor-Gortler vortices perpendicular to the main flow direction in our microfluidic device, CTCs were continuously and successfully coated with anti-epithelial cell adhesion molecule-conjugated beads. After the continuous labeling, the enlarged CTCs were perfectly trapped in a micro-filter whereas all of the leukocytes escaped. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Membrane permeability alteration of some bacterial clinical isolates by selected antihistaminics

    Directory of Open Access Journals (Sweden)

    Moustafa A El-Nakeeb

    2011-09-01

    Full Text Available Several antihistaminics possess antibacterial activity against a broad spectrum of bacteria. However, the exact mechanism of such activity was unclear. Hence, the aim of this study is to investigate their mechanism of antibacterial activity especially their effect upon the permeability of the bacterial cytoplasmic membrane. The effects of azelastine, cetirizine, cyproheptadine and diphenhydramine were studied using Gram-positive and Gram-negative multiresistant clinical isolates. Leakage of 260 and 280 nm UV-absorbing materials was detected upon treatment with the tested antihistaminics; indicative of membrane alteration. Using an artificial membrane model, cholesterol-free negatively-charged unilamellar liposomes, confirmed the effect of antihistaminics upon the membrane permeability both by showing an apparent membrane damage as observed microscopically and by detection of leakage of preloaded dye from the liposomes colorimatrically. Moreover, examination of the ultrastructure of cells treated with azelastine and cetirizine under the transmission electron microscope substantiated the detected abnormalities in the cell wall and membrane. Furthermore, the effect of pretreating certain isolates for both short and long periods with selected antihistaminics was followed by the viable count technique. Increased vulnerability towards further exposure to azelastine was observed in cells pretreated with azelastine for 2 days and those pretreated with azelastine or cetrizine for 30 days.

  19. Isolation and characterization of iron chelators from turmeric (Curcuma longa): selective metal binding by curcuminoids.

    Science.gov (United States)

    Messner, Donald J; Surrago, Christine; Fiordalisi, Celia; Chung, Wing Yin; Kowdley, Kris V

    2017-10-01

    Iron overload disorders may be treated by chelation therapy. This study describes a novel method for isolating iron chelators from complex mixtures including plant extracts. We demonstrate the one-step isolation of curcuminoids from turmeric, the medicinal food spice derived from Curcuma longa. The method uses iron-nitrilotriacetic acid (NTA)-agarose, to which curcumin binds rapidly, specifically, and reversibly. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin each bound iron-NTA-agarose with comparable affinities and a stoichiometry near 1. Analyses of binding efficiencies and purity demonstrated that curcuminoids comprise the primary iron binding compounds recovered from a crude turmeric extract. Competition of curcuminoid binding to the iron resin was used to characterize the metal binding site on curcumin and to detect iron binding by added chelators. Curcumin-Iron-NTA-agarose binding was inhibited by other metals with relative potency: (>90% inhibition) Cu2+ ~ Al3+ > Zn2+ ≥ Ca2+ ~ Mg2+ ~ Mn2+ (80% by addition of iron to the media; uptake was completely restored by desferoxamine. Ranking of metals by relative potencies for blocking curcumin uptake agreed with their relative potencies in blocking curcumin binding to iron-NTA-agarose. We conclude that curcumin can selectively bind toxic metals including iron in a physiological setting, and propose inhibition of curcumin binding to iron-NTA-agarose for iron chelator screening.

  20. Isolation of somatic Na+ currents by selective inactivation of axonal channels with a voltage prepulse.

    Science.gov (United States)

    Milescu, Lorin S; Bean, Bruce P; Smith, Jeffrey C

    2010-06-02

    We present a simple and effective method for isolating the somatic Na(+) current recorded under voltage clamp from neurons in brain slices. The principle is to convert the axon from an active compartment capable of generating uncontrolled axonal spikes into a passive structure by selectively inactivating axonal Na(+) channels. Typically, whole-cell currents from intact neurons under somatic voltage clamp contain a mixture of Na(+) current and axial current caused by escaped axonal spikes. We found that a brief prepulse to voltages near spike threshold evokes the axonal spike, which inactivates axonal but not somatic channels. A subsequent voltage step then evokes only somatic Na(+) current from electrotonically proximal sodium channels under good voltage-clamp control. Simulations using a neuron compartmental model support the idea that the prepulse effectively inactivates currents from the axon and isolates well controlled somatic currents. Na(+) currents recorded from cortical pyramidal neurons in slices, using the prepulse, were found to have voltage dependence nearly identical to that of currents recorded from acutely dissociated pyramidal neurons. In addition, studies in dissociated neurons show that the prepulse has no visible effect on the voltage dependence and kinetics of Na(+) currents elicited by the subsequent voltage step, only decreasing the amplitude of the currents by 10-20%. The technique was effective in several neuronal types in brain slices from male and female neonatal rats and mice, including raphé neurons, cortical pyramidal neurons, inferior olivary neurons, and hypoglossal motoneurons.

  1. Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish.

    Science.gov (United States)

    Lalloo, R; Ramchuran, S; Ramduth, D; Görgens, J; Gardiner, N

    2007-11-01

    To isolate, select and evaluate Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. Natural isolates obtained from mud sediment and Cyprinus carpio were purified and assessed in vitro for efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions. Based on suitability to predefined characteristics, the isolates B001, B002 and B003 were selected and evaluated in vitro in the presence of Aer. hydrophila and in a preliminary in vivo trial with C. carpio. The inhibitory effect on pathogen growth and the decrease in concentrations of waste ions was demonstrated. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, Bacillus cereus and Bacillus licheniformis, respectively. Isolate B002 did not contain the anthrax virulence plasmids pOX1, pOX2 or the B. cereus enterotoxin. Selected isolates effected synergistic reduction in pathogen load and the concentrations of waste ions in vitro and in vivo and are safe for use in ornamental aquaculture. A new approach for assessment of biological agents was demonstrated and has yielded putative isolates for development into aquaculture products.

  2. Partial Sequencing of 16S rRNA Gene of Selected Staphylococcus aureus Isolates and its Antibiotic Resistance

    Directory of Open Access Journals (Sweden)

    Harsi Dewantari Kusumaningrum

    2016-08-01

    Full Text Available The choice of primer used in 16S rRNA sequencing for identification of Staphylococcus species found in food is important. This study aimed to characterize Staphylococcus aureus isolates by partial sequencing based on 16S rRNA gene employing primers 16sF, 63F or 1387R. The isolates were isolated from milk, egg dishes and chicken dishes and selected based on the presence of sea gene that responsible for formation of enterotoxin-A. Antibiotic susceptibility of the isolates towards six antibiotics was also tested. The use of 16sF resulted generally in higher identity percentage and query coverage compared to the sequencing by 63F or 1387R. BLAST results of all isolates, sequenced by 16sF, showed 99% homology to complete genome of four S. aureus strains, with different characteristics on enterotoxin production and antibiotic resistance. Considering that all isolates were carrying sea gene, indicated by the occurence of 120 bp amplicon after PCR amplification using primer SEA1/SEA2,  the isolates were most in agreeing to S. aureus subsp. aureus ST288. This study indicated that 4 out of 8 selected isolates were resistant towards streptomycin. The 16S rRNA gene sequencing using 16sF is useful for identification of S. aureus. However, additional analysis such as PCR employing specific gene target, should give a valuable supplementary information, when specific characteristic is expected.

  3. Antibiotic content of selective culture media for isolation of Capnocytophaga species from oral polymicrobial samples.

    Science.gov (United States)

    Ehrmann, E; Jolivet-Gougeon, A; Bonnaure-Mallet, M; Fosse, T

    2013-10-01

    In oral microbiome, because of the abundance of commensal competitive flora, selective media with antibiotics are necessary for the recovery of fastidious Capnocytophaga species. The performances of six culture media (blood agar, chocolate blood agar, VCAT medium, CAPE medium, bacitracin chocolate blood agar and VK medium) were compared with literature data concerning five other media (FAA, LB, TSBV, CapR and TBBP media). To understand variable growth on selective media, the MICs of each antimicrobial agent contained in this different media (colistin, kanamycin, trimethoprim, trimethoprim-sulfamethoxazole, vancomycin, aztreonam and bacitracin) were determined for all Capnocytophaga species. Overall, VCAT medium (Columbia, 10% cooked horse blood, polyvitaminic supplement, 3·75 mg l(-1) of colistin, 1·5 mg l(-1) of trimethoprim, 1 mg l(-1) of vancomycin and 0·5 mg l(-1) of amphotericin B, Oxoid, France) was the more efficient selective medium, with regard to the detection of Capnocytophaga species from oral samples (P culture, a simple blood agar allowed the growth of all Capnocytophaga species. Nonetheless, in oral samples, because of the abundance of commensal competitive flora, selective media with antibiotics are necessary for the recovery of Capnocytophaga species. The demonstrated superiority of VCAT medium made its use essential for the optimal detection of this bacterial genus. This work showed that extreme caution should be exercised when reporting the isolation of Capnocytophaga species from oral polymicrobial samples, because the culture medium is a determining factor. © 2013 The Society for Applied Microbiology.

  4. Synthesis of Thermally Switchable Chromatographic Materials with Immobilized Ti4+ for Enrichment of Phosphopeptides by Reversible Addition Fragmentation Chain Transfer Polymerization

    Science.gov (United States)

    Wang, Di; Cao, Zhihan; Pang, Xinzhu; Deng, Yulin; Li, Bo; Dai, Rongji

    2018-01-01

    Reversible phosphorylation of proteins is one of the most crucial types of post-translational modifications (PTMs). And it shows significant work in diversified biological processes. However, the separation technology of phosphorylated peptides is still an analytical challenge in phosphoproteomics, because phosphopeptides are alway in low stoichiometry. Thus, enrichment of phosphopeptides before detection is indispensable. In this study, a novel temperature regulated separation protocol was developed. Silica@p (NIPAAm-co-IPPA)-Ti4+, a new Ti(IV)-IMAC (Immobilized Metal Affinity chromatography) materials was synthesized by reversible addition fragmentation chain transfer polymerization (RAFT). By the unique thermally responsive properties of poly(N-isopropylacrylamide) (PNIPAAm), the captured phosphorylated peptides could be released by changing temperature only without applying any other eluant which could damage the phosphopeptides. We employed isopropanol phosphonic acid (IPPA) as an IMAC ligand for the immobilization of Ti(IV) which could increase the specific adsorption of phosphopeptides. The enrichment and release properties were examined by treatment with pyridoxal 5’-phosphate (PLP) and casein phosphopeptides (CPP). Two phosphorylated compounds above have temperature-stimulated binding to Ti4+. Finally, silica@p (NIPAAm-co-IPPA)-Ti4+ was successfully employed in pretreatment of phosphopeptides in a tryptic digest of a-casein and human serum albumin (HSA). The results indicated a great potential of this new temperature-responsive material in phosphoproteomics study.

  5. Selective-differential medium for isolation and differentiation of pectinatus from other brewery microorganisms.

    Science.gov (United States)

    Lee, S Y; Moore, S E; Mabee, M S

    1981-02-01

    An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and yeast. Differentiation was established by the introduction of lead acetate into the medium, which reacted with the H(2)S liberated by Pectinatus and resulted in a blackening of the Pectinatus colonies while the other brewery organisms, when present, remained white. In combination with the Lee tube (J. E. Ogg, S. Y. Lee, and B. J. Ogg, Can. J. Microbiol. 25:987-990, 1979) and this medium, isolation of Pectinatus organisms from beer samples was accomplished with convenience and simplicity.

  6. Selected Lactic Acid-Producing Bacterial Isolates with the Capacity to Reduce Salmonella Translocation and Virulence Gene Expression in Chickens

    OpenAIRE

    Xiaojian Yang; Jennifer Brisbin; Hai Yu; Qi Wang; Fugui Yin; Yonggang Zhang; Parviz Sabour; Shayan Sharif; Joshua Gong

    2014-01-01

    BACKGROUND: Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) an...

  7. In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Højlund, Kurt; Bowen, Benjamin P; Hwang, Hyonson

    2009-01-01

    Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin resistant muscle using...... volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including...... phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy...

  8. Selection and characterization of Argentine isolates of Trichoderma harzianum for effective biocontrol of Septoria leaf blotch of wheat.

    Science.gov (United States)

    Stocco, Marina C; Mónaco, Cecilia I; Abramoff, Cecilia; Lampugnani, Gladys; Salerno, Graciela; Kripelz, Natalia; Cordo, Cristina A; Consolo, Verónica F

    2016-03-01

    Species of the genus Trichoderma are economically important as biocontrol agents, serving as a potential alternative to chemical control. The applicability of Trichoderma isolates to different ecozones will depend on the behavior of the strains selected from each zone. The present study was undertaken to isolate biocontrol populations of Trichoderma spp. from the Argentine wheat regions and to select and characterize the best strains of Trichoderma harzianum by means of molecular techniques. A total of 84 out of the 240 strains of Trichoderma were able to reduce the disease severity of the leaf blotch of wheat. Thirty-seven strains were selected for the reduction equal to or greater than 50% of the severity, compared with the control. The percentage values of reduction of the pycnidial coverage ranged between 45 and 80%. The same last strains were confirmed as T. harzianum by polymerase chain reaction amplification of internal transcribed spacers, followed by sequencing. Inter-simple sequence repeat was used to examine the genetic variability among isolates. This resulted in a total of 132 bands. Further numerical analysis revealed 19 haplotypes, grouped in three clusters (I, II, III). Shared strains, with different geographical origins and isolated in different years, were observed within each cluster. The origin of the isolates and the genetic group were partially related. All isolates from Paraná were in cluster I, all isolates from Lobería were in cluster II, and all isolates from Pergamino and Santa Fe were in cluster III. Our results suggest that the 37 native strains of T. harzianum are important in biocontrol programs and could be advantageous for the preparation of biopesticides adapted to the agroecological conditions of wheat culture.

  9. Angoline: a selective IL-6/STAT3 signaling pathway inhibitor isolated from Zanthoxylum nitidum.

    Science.gov (United States)

    Liu, Jiawei; Zhang, Qing; Ye, Yushan; Li, Wuguo; Qiu, Junxin; Liu, Jingli; Zhan, Ruoting; Chen, Weiwen; Yu, Qiang

    2014-01-01

    STAT3 signaling pathway is an important target for human cancer therapy. Thus, the identification of small-molecules that target STAT3 signaling will be of great interests in the development of anticancer agents. The aim of this study was to identify novel inhibitors of STAT3 pathway from the roots of Zanthoxylum nitidum (Roxb.) DC. The bioassay-guided fractionation of MeOH extract of Z. nitidum using a STAT3-responsive gene reporter assay led to the isolation of angoline (1) as a potent and selective inhibitor of the STAT3 signaling pathway (IC50=11.56 μM). Angoline inhibited STAT3 phosphorylation and its target gene expression and consequently induced growth inhibition of human cancer cells with constitutively activated STAT3 (IC50=3.14-4.72 μM). This work provided a novel lead for the development of anti-cancer agents targeting the STAT3 signaling pathway. Copyright © 2014 Elsevier GmbH. All rights reserved.

  10. Using selective chromogenic plates to optimize isolation of group B Streptococcus in pregnant women

    Directory of Open Access Journals (Sweden)

    Romano Mattei

    2014-03-01

    Full Text Available Group B Streptococcus (GBS remains the leading cause of severe bacterial infections (sepsis, meningitis, pneumonia in neonates. We compared the detection of GBS from recto-vaginal swabs on blood agar and two chromogenic media and evaluated their antibiotic susceptibility. A total of 1351 swabs were taken from pregnant women at 35-37 weeks of gestation. Following enrichment in Todd Hewitt broth + nalidixic acid and colistin, the samples were plated on Columbia CNA agar (CNA, chromID Strepto B agar (STRB and Granada Agar (GRAN, respectively. GBS were found in 22.4% of recto-vaginal swabs from pregnant women. Sensitivity, specificity, positive and negative predictive values of GBS detection were 88%, 88%, 81% and 96% for CNA, 99%, 97%, 90% and 99% for STRB and 94%, 99%, 98% e 99% for GRAN; Cohen’s k index concordances for CNA, STREB and GRAN were 0.68, 0.92 and 0.96, respectively. All isolates were susceptible to penicillin, whereas resistances of erythromycin and clindamycin were 40% and 42%, respectively. To conclude, selective broth enrichment combined with chromogenic plates is recommended for GBS screening in pregnant women.

  11. The Use of Variable Q1 Isolation Windows Improves Selectivity in LC-SWATH-MS Acquisition.

    Science.gov (United States)

    Zhang, Ying; Bilbao, Aivett; Bruderer, Tobias; Luban, Jeremy; Strambio-De-Castillia, Caterina; Lisacek, Frédérique; Hopfgartner, Gérard; Varesio, Emmanuel

    2015-10-02

    As tryptic peptides and metabolites are not equally distributed along the mass range, the probability of cross fragment ion interference is higher in certain windows when fixed Q1 SWATH windows are applied. We evaluated the benefits of utilizing variable Q1 SWATH windows with regards to selectivity improvement. Variable windows based on equalizing the distribution of either the precursor ion population (PIP) or the total ion current (TIC) within each window were generated by an in-house software, swathTUNER. These two variable Q1 SWATH window strategies outperformed, with respect to quantification and identification, the basic approach using a fixed window width (FIX) for proteomic profiling of human monocyte-derived dendritic cells (MDDCs). Thus, 13.8 and 8.4% additional peptide precursors, which resulted in 13.1 and 10.0% more proteins, were confidently identified by SWATH using the strategy PIP and TIC, respectively, in the MDDC proteomic sample. On the basis of the spectral library purity score, some improvement warranted by variable Q1 windows was also observed, albeit to a lesser extent, in the metabolomic profiling of human urine. We show that the novel concept of "scheduled SWATH" proposed here, which incorporates (i) variable isolation windows and (ii) precursor retention time segmentation further improves both peptide and metabolite identifications.

  12. An isolation and confinement facility for the selection of astronaut candidates.

    Science.gov (United States)

    Inoue, Natsuhiko; Tachibana, Shoichi

    2013-08-01

    In 2008, JAXA conducted its fifth astronaut candidate recruitment. The final stage involved 10 of 963 applicants undergoing the first and second examination stages based on physical checks, written tests, and interviews. The third (final) stage focused on evaluating the behavioral attitudes required for ISS crewmembers. Since it is difficult to assess these attitudes by paper testing or interview alone, JAXA has established an environment in which the assessors could observe the behavior of examinees on a continuous basis. The 10 examinees stayed in the isolation and confinement facility for 1 wk. During their stay, several group and personal tasks were assigned along with predetermined daily schedules covering aspects of leadership, teamwork ability, productivity (performance), and so on. Subsequently, psychologists, psychiatrists, and JAXA officials/managers experienced in practical human space development evaluated them. During the confinement, no examinees showed any excessive character traits or psychopathologic behavior. Repeated observations led to a convergence of opinions among the experts. Three examinees ultimately passed this assessment and were assigned as new Japanese astronaut candidates. This unique assessment enabled the assessors to conduct longitudinal evaluations, evaluations based on observing detailed behavior, and easy control of test conditions and safety. Although "selection of the right stuff" may differ among each space agency, this method offers the unique advantage of allowing experts in various fields to evaluate the competencies of examinees both synthetically and longitudinally.

  13. Antibacterial Action of Jineol Isolated from Scolopendra subspinipes mutilans against Selected Foodborne Pathogens.

    Science.gov (United States)

    Bajpai, Vivek K; Shukla, Shruti; Paek, Woon K; Lim, Jeongheui; Kumar, Pradeep; Na, MinKyun

    2017-01-01

    This study was undertaken to assess the antibacterial potential of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans against selected foodborne pathogens Escherichia coli O157:H7 and Staphylococcus aureus KCTC-1621. Jineol at the tested concentration (50 μL; corresponding to 250 μg/disk) exhibited significant antibacterial effects as a diameter of inhibition zones (11.6-13.6 mm), along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration values found in the range of (62.5-125 μg/mL) and (125-250 μg/mL), respectively. Jineol also exhibited significant antibacterial effects as confirmed by the reduction in bacterial cell viabilities, increasing release of potassium (K(+)) ions (650 and 700 mmole/L) and 260 nm materials (optical density: 2.98-3.12) against both the tested pathogens, E. coli O157:H7 and S. aureus KCTC-1621, respectively. Moreover, changes in the cell wall morphology of E. coli O157:H7 and S. aureus KCTC-1621 cells treated with jineol at MIC further confirmed its inhibitory potential against the tested pathogens, suggesting its role as an effective antimicrobial to control foodborne pathogens.

  14. Selected, annotated bibliography of studies relevant to the isolation of nuclear wastes. [705 references

    Energy Technology Data Exchange (ETDEWEB)

    Hyder, L.K.; Fore, C.S.; Vaughan, N.D.; Faust, R.A.

    1980-09-01

    This annotated bibliography of 705 references represents the first in a series to be published by the Ecological Sciences Information Center containing scientific, technical, economic, and regulatory information relevant to nuclear waste isolation. Most references discuss deep geologic disposal, with fewer studies of deep seabed disposal; space disposal is also included. The publication covers both domestic and foreign literature for the period 1954 to 1980. Major chapters selected are Chemical and Physical Aspects; Container Design and Performance; Disposal Site; Envirnmental Transport; General Studies and Reviews; Geology, Hydrology and Site Resources; Regulatory and Economic Aspects; Repository Design and Engineering; Transportation Technology; Waste Production; and Waste Treatment. Specialized data fields have been incorporated to improve the ease and accuracy of locating pertinent references. Specific radionuclides for which data are presented are listed in the Measured Radionuclides field, and specific parameters which affect the migration of these radionuclides are presented in the Measured Parameters field. The references within each chapter are arranged alphabetically by leading author, corporate affiliation, or title of the document. When the author is not given, the corporate affiliation appears first. If these two levels of authorship are not given, the title of the document is used as the identifying level. Indexes are provided for author(s), keywords, subject category, title, geographic location, measured parameters, measured radionuclides, and publication description.

  15. Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins.

    Science.gov (United States)

    Rafiee, Mahmoud-Reza; Girardot, Charles; Sigismondo, Gianluca; Krijgsveld, Jeroen

    2016-11-03

    Maintenance of pluripotency is regulated by a network of transcription factors coordinated by Oct4, Sox2, and Nanog (OSN), yet a systematic investigation of the composition and dynamics of the OSN protein network specifically on chromatin is still missing. Here we have developed a method combining ChIP with selective isolation of chromatin-associated proteins (SICAP) followed by mass spectrometry to identify chromatin-bound partners of a protein of interest. ChIP-SICAP in mouse embryonic stem cells (ESCs) identified over 400 proteins associating with OSN, including several whose interaction depends on the pluripotent state. Trim24, a previously unrecognized protein in the network, converges with OSN on multiple enhancers and suppresses the expression of developmental genes while activating cell cycle genes. Consistently, Trim24 significantly improved efficiency of cellular reprogramming, demonstrating its direct functionality in establishing pluripotency. Collectively, ChIP-SICAP provides a powerful tool to decode chromatin protein composition, further enhanced by its integrative capacity to perform ChIP-seq. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Characterization of Enterococcus Isolates Colonizing the Intestinal Tract of Intensive Care Unit Patients Receiving Selective Digestive Decontamination

    Science.gov (United States)

    Bello Gonzalez, Teresita d. J.; Pham, Phu; Top, Janetta; Willems, Rob J. L.; van Schaik, Willem; van Passel, Mark W. J.; Smidt, Hauke

    2017-01-01

    Enterococci have emerged as important opportunistic pathogens in intensive care units (ICUs). In this study, enterococcal population size and Enterococcus isolates colonizing the intestinal tract of ICU patients receiving Selective Digestive Decontamination (SDD) were investigated. All nine patients included in the study showed substantial shifts in the enterococcal 16S rRNA gene copy number in the gut microbiota during the hospitalization period. Furthermore, 41 Enterococcus spp. strains were isolated and characterized from these patients at different time points during and after ICU hospitalization, including E. faecalis (n = 13), E. faecium (n = 23), and five isolates that could not unequivocally assigned to a specific species (E. sp. n = 5) Multi locus sequence typing revealed a high prevalence of ST 6 in E. faecalis isolates (46%) and ST 117 in E. faecium (52%). Furthermore, antibiotic resistance phenotypes, including macrolide and vancomycin resistance, as well as virulence factor-encoding genes [asa1, esp-fm, esp-fs, hyl, and cyl (B)] were investigated in all isolates. Resistance to ampicillin and tetracycline was observed in 25 (61%) and 19 (46%) isolates, respectively. Furthermore, 30 out of 41 isolates harbored the erm (B) gene, mainly present in E. faecium isolates (78%). The most prevalent virulence genes were asa1 in E. faecalis (54%) and esp (esp-fm, 74%; esp-fs, 39%). Six out of nine patients developed nosocomial enterococcal infections, however, corresponding clinical isolates were unfortunately not available for further analysis. Our results show that multiple Enterococcus species, carrying several antibiotic resistance and virulence genes, occurred simultaneously in patients receiving SDD therapy, with varying prevalence dynamics over time. Furthermore, simultaneous presence and/or replacement of E. faecium STs was observed-, reinforcing the importance of screening multiple isolates to comprehensively characterize enterococcal diversity in ICU

  17. Selected fault testing of electronic isolation devices used in nuclear power plant operation

    Energy Technology Data Exchange (ETDEWEB)

    Villaran, M.; Hillman, K.; Taylor, J.; Lara, J.; Wilhelm, W. [Brookhaven National Lab., Upton, NY (United States)

    1994-05-01

    Electronic isolation devices are used in nuclear power plants to provide electrical separation between safety and non-safety circuits and systems. Major fault testing in an earlier program indicated that some energy may pass through an isolation device when a fault at the maximum credible potential is applied in the transverse mode to its output terminals. During subsequent field qualification testing of isolators, concerns were raised that the worst case fault, that is, the maximum credible fault (MCF), may not occur with a fault at the maximum credible potential, but rather at some lower potential. The present test program investigates whether problems can arise when fault levels up to the MCF potential are applied to the output terminals of an isolator. The fault energy passed through an isolated device during a fault was measured to determine whether the levels are great enough to potentially damage or degrade performance of equipment on the input (Class 1E) side of the isolator.

  18. Effect of casein phosphopeptide - amorphous calcium phosphate containing chewing gum on salivary concentration of calcium and phosphorus: An in-vivo study

    Directory of Open Access Journals (Sweden)

    B P Santhosh

    2012-01-01

    Full Text Available Aim: Caries clinical trials of sugar-free chewing gum have shown that the gum is noncariogenic and in fact has anticariogenic effect through the stimulation of saliva. Sugar-free gums, therefore, may be an excellent delivery vehicle for safe and effective additive, capable of promoting enamel remineralization. Casein phosphopeptide - amorphous calcium phosphate (CPP-ACP nanocomplexes incorporated into sugar-free chewing gum have shown to remineralize enamel subsurface lesions in situ. So this study was conducted to evaluate the effect of CPP-ACP containing sugar-free chewing gum on salivary concentration of calcium and phosphorous. Materials and Methods : Unstimulated saliva from each 24 selected subjects was collected. Then each subject was given two pellets of chewing gum containing CPP-ACP and asked to chew for a period of 20 min, after which saliva samples were collected from each individual. Once all the samples were collected they were assessed for calcium and phosphorous concentration using affiliated reagent kits and photometer. Statistical Analysis Used: Data obtained were analyzed using student′s paired t test. Results: Significant difference was found in the calcium and phosphorus concentration of saliva before and after chewing CPP-ACP containing chewing gum. Conclusions: Chewing of CPP-ACP containing chewing gum showed a significant increase in the salivary concentration of calcium for a prolonged period of time hence it may help in the remineralization of tooth surfaces.

  19. Maize glutamine synthetase cDNAs: isolation by direct genetic selection in Escherichia coli.

    Science.gov (United States)

    Snustad, D P; Hunsperger, J P; Chereskin, B M; Messing, J

    1988-12-01

    Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.

  20. Selection of Leuconostoc strains isolated from artisanal Serrano Catarinense cheese for use as adjuncts in cheese manufacture.

    Science.gov (United States)

    Seixas, Felipe Nael; Rios, Edson Antônio; Martinez de Oliveira, André Luiz; Beloti, Vanerli; Poveda, Justa Maria

    2018-01-24

    Serrano Catarinense cheese is a raw bovine milk cheese produced in the region of Santa Catarina, Brazil. Twelve representative strains of Leuconostoc isolated from 20 samples of this artisanal cheese were selected and submitted for evaluation of the acidifying, proteolytic, autolytic, aminopeptidase and lipolytic activities, NaCl and acid resistance, production of dextran and biogenic amines and antimicrobial activity. The aim was to genetically and technologically characterize the Leuconostoc strains in order to use them in mixed starter cultures for cheese manufacture. L. mesenteroides subsp. mesenteroides was the species that accounted for the largest proportion of isolates of Leuconostoc genus. Two leuconostocs isolates stood out in the acidifying activity, with reduction in pH of 1.12 and 1.04 units. The isolates showed low proteolytic and autolytic activity. Most of the isolates were dextran producers, presented good resistance to the salt and pH conditions of the cheese, showed antimicrobial activity against cheese pathogen bacteria and none of them produced biogenic amines. These results allowed the selection of five strains (UEL 04, UEL 12, UEL 18, UEL 21 and UEL 28) as good candidates for use as adjunct cultures for cheese manufacture. This article is protected by copyright. All rights reserved.

  1. Selection and characterization of Beauveria spp. isolates to control the broad mite Polyphagotarsonemus latus (Banks, 1904 (Acari: Tarsonemidae

    Directory of Open Access Journals (Sweden)

    C. C. Martins

    Full Text Available Abstract This study was performed under laboratory conditions to identify isolates of the fungus Beauveria spp. that can control Polyphagotarsonemus latus in the greenhouse and field. Thirty Beauveria spp. isolates were tested by spraying 1 mL conidia (1 × 108 conidia/mL on pepper leaf discs containing 15 mites. Evaluations were performed on the 3rd and 6th day post application by counting the number of dead mites. Vegetative growth and conidial production were measured from the selected isolates, and bioassays were conducted in the greenhouse on bean seedlings in plastic pots. The isolate Unioeste 53 was selected, and a conidial suspension (1 × 108 conidia/mL was applied with a backpack sprayer. The evaluation consisted of pre- and post-treatment counts of the number of live mites on ten leaflets in both the plots treated with the fungus and control plots, and the same procedure was followed for the field experiment. In the laboratory, the Unioeste 53 isolate resulted in total and confirmed mortality rates of 70% and 57.7%, respectively. In the greenhouse, the population decreased by 76.71% by the 16th day after application. In the field, the population decreased by 66% by the 12th day after application, demonstrating the potential of this fungus for mite management.

  2. Selection, fitness, and control of grape isolates of Botrytis cinerea variably sensitive to fenhexamid

    Science.gov (United States)

    Of 683 Botrytis cinerea isolates collected from a fungicide-trial vineyard, 31 were classified as putatively resistant to fenhexamid (EC50 =0.1 ug/ml). For the resistant isolates that survived and sporulated in culture, colony expansion and conidial germination frequency was significantly reduced re...

  3. Simultaneous enrichment of cysteine-containing peptides and phosphopeptides using a cysteine-specific phosphonate adaptable tag (CysPAT) in combination with titanium dioxide (TiO2) chromatography

    DEFF Research Database (Denmark)

    Huang, Honggang; Pedersen, Martin Haar; Ibañez-Vea, Maria

    2016-01-01

    to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from...... Hela cells, achieving high specificity and enrichment efficiency. In particular, for Cys proteome analysis, the method led to the identification of 7509 unique Cys peptides from 500 μg of HeLa cell lysate starting material. Furthermore, the method was developed to simultaneously enrich Cys peptides...... and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 μg starting...

  4. OPTIMIZATION OF IMMOBILIZED METAL ION AFFINITY CHROMATOGRAPHY FOR PHOSPHOPEPTIDE ENRICHMENT  PRIOR TO MASS SPECTROMETRY

    DEFF Research Database (Denmark)

    Ye, Juanying; Zhang, Xumin; Young, Clifford

    simple procedures.     Methods Tryptic digests of standard phosphoproteins (bovine α,β- casein) and 3 non-phosphoproteins (bovine serum albumin, bovine β-lactoglobulin, and bovine carbonic anhydrase) with different ratios (1:50, 1:200, 1:500, 1:1000) were used for Fe(III)-IMAC (Qiagen Ni-NTA) enrichment...... experiments. Various compositions of loading buffer, washing buffer and elution buffer were tested and the results were analyzed by MALDI-TOF MS (Bruker UltraFlex TOF/TOF mass spectrometer). Application of this method to phosphoproteomic analysis of Arabidopsis thaliana membrane proteins is in progress...... that highly selective enrichment can be achieved by the improved method even when using highly diluted phosphopeptide samples in a background of peptides (1:1000). The improved method also proved to be advantageous in minimizing sample loss. The explanation of the improvement might result from the enhanced...

  5. Simultaneous enrichment of cysteine-containing peptides and phosphopeptides using a cysteine-specific phosphonate adaptable tag (CysPAT) in combination with titanium dioxide (TiO2) chromatography

    DEFF Research Database (Denmark)

    Huang, Honggang; Pedersen, Martin Haar; Ibañez-Vea, Maria

    2016-01-01

    to selectively label cysteine-containing peptides (Cys peptides) followed by their enrichment with titanium dioxide (TiO2) and subsequent mass spectrometric analysis. The CysPAT strategy was developed using a synthetic peptide, a standard protein and subsequently the strategy was applied to protein lysates from...... and phosphorylated peptides. This strategy was applied to SILAC labeled Hela cells subjected to 5 min epidermal growth factor (EGF) stimulation. In total, 10440 unique reversibly modified Cys peptides (3855 proteins) and 7339 unique phosphopeptides (2234 proteins) were simultaneously identified from 250 μg starting...... material. Significant regulation was observed in both phosphorylation and reversible Cys modification of proteins involved in EGFR signaling. Our data indicates that EGF stimulation can activate the well-known phosphorylation of EGFR and downstream signaling molecules, such as mitogen-activated protein...

  6. Isolation and selection of fluorescent pseudomonads based on multiple plant growth promotion traits and siderotyping

    Directory of Open Access Journals (Sweden)

    Jayamohan Subramanian

    2014-09-01

    Full Text Available Fluorescent pseudomonads, acclaimed plant associated bacterial group, are well-known plant growth promoting-biocontrol agents in rhizosphere arena. In this study, 144 fluorescent pseudomonad isolates from rhizosphere soil samples were screened with King's medium B supplemented with 8-hydroxyquinoline (8-HQ chelator and comprehensively profiled for plant growth promotion viz., production of indole acetic acid (IAA, siderophore, ammonia, hydrogen cyanide, motility, phosphate solubilization, root growth promotion, and biofilm forming ability, along with two known control strains of pseudomonads. Iron and IAA regulated secondary metabolite siderophore production were investigated quantitatively. All isolates were positive for ammonia production and motility; 46% isolates were positive for hydrogen cyanide, 44% shown positivity for phosphate solubilization, and 40% isolates for siderophore production. Siderotyping showed production of hydroxamate type of siderophores which are known to be more efficient biocontrol agents. All isolates stimulated root growth to varying extent and had potentiality to form biofilms, a critical constituent for survival on different environments. Forty-two isolates of pseudomonads showed antagonistic behavior against the deleterious fungal pathogen Fusarium oxysporum (MTCC1755. Based on the above observations and statistical analysis, 11 isolates were shortlisted for further scrutiny. The study of biogeographic correlation and secondary metabolite profiling in association with plant growth promotion focalizes significant assessment on the behavior and antagonistic action, which probably brings out a competent biocontrol agent in a sustainable eco-friendly dimension.

  7. Comparison of Several Selective Media for Isolation and Differentiation of Coagulase-Positive Strains of Staphylococcus aureus1

    Science.gov (United States)

    McDivitt, Maxine E.; Topp, Eleanor B.

    1964-01-01

    Six coagulase-positive strains of Staphylococcus aureus which had been cultivated in Brain Heart Infusion broth, milk, and brine were plated on seven isolation media. A significant difference in the growth patterns of the individual strains was found as well as a significant effect resulting from the previous cultivation history before plating. Brine and, to a lesser extent, milk were found to reduce maximal cell concentrations attained, but strains grown in brine and milk showed greater ability to withstand the selective action of the isolation media. Fibrinogen applied to the surface of five of the media allowed the formation of characteristic halos by coagulase-positive strains of S. aureus. Only half of the strains studied produced a zone of precipitation on SM110-Egg Yolk agar. The isolation medium containing cycloheximide and a high level of polymxin B was most inhibitory to the organisms. PMID:14131367

  8. Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones.

    Directory of Open Access Journals (Sweden)

    Fortunata Iacopino

    Full Text Available Cancer stem cells (CSC were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11 were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2; proliferation; differentiation capability (determined by the expression of GalC, βIII-Tubulin and GFAP; Ca(2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (βIII-Tubulin and GFAP were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and βIII-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca(2+-channels but they exhibited increased intracellular Ca(2+ levels in response to ATP. These Ca(2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca(2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches.

  9. Isolation and Fatty Acid Profile of Selected Microalgae Strains from the Red Sea for Biofuel Production

    OpenAIRE

    Hugo Pereira; Luísa Barreira; Luísa Custódio; Salman Alrokayan; Fouzi Mouffouk; João Varela; Khalid M. Abu-Salah; Radhouan Ben-Hamadou

    2013-01-01

    The isolation of lipid-rich autochthonous strains of microalgae is a crucial stage for the development of a microalgae-based biofuel production plant, as these microalgae already have the necessary adaptations to withstand competition, predation and the temperatures observed at each production site. This is particularly important in extreme climates such as in Saudi Arabia. Resorting to fluorescence activated cell sorting (FACS) we screened for and isolated several microalgal strains from sam...

  10. Antibiogram profie of Pseudomonas aeruginosa isolated from some selected hospital environmental drains

    Directory of Open Access Journals (Sweden)

    Ekioba Olohigbe Imanah

    2017-10-01

    Full Text Available Objective: To isolate, identify and characterize Pseudomonas aeruginosa (P. aeruginosa from hospital drains using culture-based and PCR methods. Methods: Wastewater samples were obtained from hospital drains between August and October, 2015, using standard culture-based methods for isolation of P. aeruginosa. The isolates were further confirmed by specie-specific primer sets. Antimicrobial susceptibility testing of the isolates was conducted using the Kirby-Bauer disc diffusion method. Results: The mean P. aeruginosa population densities were expressed ranging between (1.7 × 105 ± 0.1 and (6.1 × 105 ± 0.2 CFU/mL. The antimicrobial susceptibility profile of the P. aeruginosa isolates revealed that all the isolates 96/96 (100% were resistant to penicillins (amoxicillin and cloxacillin as well as penicillin/β-lactamase inhibitor (augmentin while sensitivity was observed in carbapenems [imipenem 96/96 (100%]. The multiple antimicrobial resistance index of the P. aeruginosa to the antimicrobials used ranged from 0.33 to 0.89 while the multidrug resistant profile revealed resistance to augmentin, amoxicillin, cloxacillin. Conclusions: The present study reveals that hospital drains are potential reservoirs of multiple antibiotic resistances of P. aeruginosa.

  11. Isolation of saprophytic Leptospira spp. from a selected environmental water source of Argentina.

    Science.gov (United States)

    Scialfa, Exequiel; Grune, Sylvia; Brihuega, Bibiana; Aguirre, Pablo; Rivero, Marina

    2017-11-29

    Ten Leptospira spp. strains were isolated from water samples from Nievas stream, Olavarría, Buenos Aires province (Argentina). The isolates showed the typical motility and morphology of the genus Leptospira under dark field microscopy, developing in liquid EMJH medium after eight days of incubation at 13°C and 30°C. All isolates were negative by the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Molecular identification by 16S rRNA gene sequencing identified all isolates as nonpathogenic leptospires. Four isolates showed a genetic profile identical to that of the reference strain Leptospira biflexa serovar Patoc, and six isolates revealed sequence similarities within the 97-98% range, closely related to Leptospira yanagawae and Leptospira meyeri, respectively. Strains ScialfaASA42, ScialfaASA45, ScialfaASA44, ScialfaASA47, ScialfaASA49, ScialfaASA50 and ScialfaASA51 possibly represent a novel species of the genus Leptospira. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  12. Isolation and selection of plant growth-promoting bacteria associated with sugarcane

    Directory of Open Access Journals (Sweden)

    Ariana Alves Rodrigues

    2016-06-01

    Full Text Available Microorganisms play a vital role in maintaining soil fertility and plant health. They can act as biofertilizers and increase the resistance to biotic and abiotic stress. This study aimed at isolating and characterizing plant growth-promoting bacteria associated with sugarcane, as well as assessing their ability to promote plant growth. Endophytic bacteria from leaf, stem, root and rhizosphere were isolated from the RB 867515 commercial sugarcane variety and screened for indole acetic acid (IAA production, ability to solubilize phosphate, fix nitrogen and produce hydrogen cyanide (HCN, ammonia and the enzymes pectinase, cellulase and chitinase. A total of 136 bacteria were isolated, with 83 of them presenting some plant growth mechanism: 47 % phosphate solubilizers, 26 % nitrogen fixers and 57 % producing IAA, 0.7 % HCN and chitinase, 45 % ammonia, 30 % cellulose and 8 % pectinase. The seven best isolates were tested for their ability to promote plant growth in maize. The isolates tested for plant growth promotion belong to the Enterobacteriaceae family and the Klebsiella, Enterobacter and Pantoea genera. Five isolates promoted plant growth in greenhouse experiments, showing potential as biofertilizers.

  13. Adjustment of pH of enrichment media might improve selective isolation of MRSA from pig samples

    DEFF Research Database (Denmark)

    Cavaco, Lina; Agersø, Yvonne; Mordhorst, Hanne

    2011-01-01

    Methicillin resistant Staphylococcus aureus (MRSA) have emerged in livestock in several countries worldwide in recent years. MRSA may colonise in low numbers which makes both epidemiological studies and the implementation of control programmes difficult. Methods for selective isolation of MRSA from...... animal samples have been developed. However, obtaining sufficient sensitivity has been a challenge. Staphylococcus aureus is normally found on the skin, surviving and growing under extreme conditions: dry environment with high salt and low pH. In the selective isolation so far used high salt...... pig swabs. Initially a total of seven strains, including: two MRSA, two enterococci, two CNS one Aerococcus viridans and one Proteus spp. strains, were tested for growth in Mueller Hinton II broth with pH ranging from 4 to 5.5 and salt addition of 4% to 7%. In the next step, these strains were tested...

  14. [Selective decontamination of the intestine and total gnotobiologic isolation in treatment of mice with acute radiation sickness].

    Science.gov (United States)

    Romanchuk, L A; Bush, V; Korshunov, V M

    1992-01-01

    Comparative study of ciprofloxacin, pefloxacin and amikacin showed that ciprofloxacin and pefloxacin had a selective action on the intestinal microflora of conventional mice. Amikacin induced inhibition not only of the opportunistic bacteria but also of the representatives of the indigenous microflora. The use of the drugs in combination with total gnotobiological isolation in treatment of experimental radiation sickness provided an increase in survival of the irradiated animals, the more so as ciprofloxacin was used.

  15. Determination of the influence of substrate concentration on enzyme selectivity using whey protein Isolate and Bacillus licheniformis protease.

    Science.gov (United States)

    Butré, Claire I; Sforza, Stefano; Gruppen, Harry; Wierenga, Peter A

    2014-10-22

    Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each cleavage site in the protein. It was determined from the identification and quantification of the peptides present in the hydrolysates. Solutions of 0.1-10% (w/v) whey protein isolate (WPI) were hydrolyzed by Bacillus licheniformis protease at constant enzyme-to-substrate ratio. The cleavage sites were divided into five groups, from very high (>10%) to very low selectivity (concentrations. This finding shows that both the rate of hydrolysis and the enzyme selectivity were influenced by the substrate concentration.

  16. Super-SILAC mix coupled with SIM/AIMS assays for targeted verification of phosphopeptides discovered in a large-scale phosphoproteome analysis of hepatocellular carcinoma.

    Science.gov (United States)

    Lin, Yu-Tsun; Chien, Kun-Yi; Wu, Chia-Chun; Chang, Wen-Yu; Chu, Lichieh Julie; Chen, Min-Chi; Yeh, Chau-Ting; Yu, Jau-Song

    2017-03-22

    Plentiful studies have established a close association between aberrant phosphorylation and hepatocellular carcinoma (HCC). Here, we applied a quantitative phosphoproteomics platform combining dimethylation labeling and online 3D strong cation exchange chromatography (SCX)-titanium oxide (TiO2)/RP-LTQ-Orbitrap to compare phosphoproteomes between three pairs of HCC tissues and non-tumor counterparts. This analysis yielded 7868 quantifiable phosphopeptides and numerous up- or down-regulated candidates. Increased phosphorylation of LMNA and NIPA was confirmed using specific antibodies. To expand our verification capability, we evaluated the use of LTQ-Orbitrap run in SIM/Accurate inclusion mass screening (AIMS) mode with a super-SILAC mixture as an internal standard to quantify a subset of phosphopeptide candidates in HCC tissue samples. In sample I used for discovery experiment, we successfully quantified 32 (in SIM mode) and 30 (in AIMS mode) phosphopeptides with median coefficients of variation (CVs) of 7.5% and 8.3%, respectively. When the assay was applied to other three pairs of HCC specimens for verification experiment, 40 target phosphopeptides were quantified reliably (~7.5% CV), and more than half of them were differentially expressed between tumor and adjacent non-tumor tissues. Collectively, these results indicate the feasibility of using super-SILAC mix-SIM/AIMS assays for targeted verification of phosphopeptides discovered by large-scale phosphoproteome analyses of HCC specimens. In this study, we developed a strategy for conducting both discovery and targeted verification of deregulated phosphoproteins in HCC tissue specimens on LTQ-Orbitrap. This strategy allowed us to generate a quantitative HCC tissue phosphoproteome dataset containing significantly deregulated phosphoproteins that represents a valuable resource for the identification of potential HCC biomarkers and/or therapeutic targets. Furthermore, our proof-of-concept experiments demonstrated

  17. Protective effect of casein phosphopeptide-amorphous calcium phosphate on enamel erosion: Atomic force microscopy studies.

    Science.gov (United States)

    Ceci, Matteo; Mirando, Maria; Beltrami, Riccardo; Chiesa, Marco; Poggio, Claudio

    2015-01-01

    The aim of this study was to investigate the in vitro effect of a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste (GC Tooth Mousse- TM, GC Corporation, Tokyo, Japan) on preventing enamel erosion, by using Atomic Force Microscopy (AFM). 30 human incisors, were equally assigned to 6 groups: intact enamel, enamel + soft drink, enamel + TM, enamel + TM + soft drink, enamel + soft drink + TM, enamel + soft drink + TM + soft drink. Specimens were observed through atomic force microscopy (AFM). The most common topographical parameters were determined, such as the surface roughness (Rrms ). The use of soft drink on intact enamel has roughened the surface of the sample. The application of the CPP-ACP paste on non-treated enamel made the surface smoother. A significant decrease in roughness was seen after remineralization with CPP-ACP paste. Significant differences were recorded when comparing softened enamel with softened enamel remineralized with CPP-ACP paste. Comparing eroded enamel with demineralized/remineralized specimens, the application of a CPP-ACP paste leads to a significant reduction in roughness values. AFM images of enamel surface treated with CPP-ACP resulted in less morphological changes of the tooth substrate when compared with the only eroded enamel surface morphology; thus, indicating that CPP-ACP paste promoted remineralization. Specimens' surface roughness remained similar regardless that the protective agent is used before or after exposure to coke or between two demineralizing cycles. The results confirmed the effectiveness of the CPP-ACP paste on preventing enamel erosion produced by soft drinks. © Wiley Periodicals, Inc.

  18. Protective potential of casein phosphopeptide amorphous calcium phosphate containing paste on enamel surfaces.

    Science.gov (United States)

    Somasundaram, Padmini; Vimala, N; Mandke, Lalita Gauri

    2013-03-01

    Dental caries remains the most common dental disease facing mankind. Prevention of initiation and interruption in progression of early lesions are the desirable modes of caries management. There is a scope for agents, which may be used to enhance anti - caries activity. This need has redirected research to develop novel preventive agents that can act as an adjunct to fluoride or independent of it. Casein Phosphopeptide - Amorphous Calcium Phosphate (CPP-ACP) is one such agent that has been proposed to have anti cariogenic properties. The purpose of this in vitro study was to evaluate the effect of paste containing CPP-ACP, MI Paste, on enamel remineralization. This study consisted of 30 samples embedded in orthodontic resin with either the buccal or lingual surface exposed. The samples were assigned to either a CPP-ACP containing paste; Fluoridated toothpaste; or a control group. The groups were then subjected to cycling in a demineralizing solution and a remineralizing solution. Groups II and III received prior application of MI paste and Fluoridated toothpaste respectively followed by cycling in a demineralizing solution and a remineralizing solution. Following 14 days of cycling, the samples were sectioned and examined using confocal microscopy. The lesion depth, were evaluated. Image Proplus software was used to analyze the images. The values were statistically evaluated using one - way ANOVA and Scheffe's Test. Within the limitations of the study it was concluded that enamel surfaces treated with the CPP-ACP paste exhibited the least lesion depths followed by the enamel surfaces treated with the fluoridated tooth paste and control group respectively.

  19. Antimicrobial testing of selected fluoroquinolones against Pseudomonas aeruginosa isolated from canine otitis.

    Science.gov (United States)

    McKay, Lindsay; Rose, Crystal D Schuman; Matousek, Jennifer L; Schmeitzel, Lynn S; Gibson, Nicole M; Gaskin, Jack M

    2007-01-01

    A total of 100 Pseudomonas aeruginosa (P. aeruginosa) isolates were collected over a 1.5-year period from cases of canine otitis. Sensitivities to enrofloxacin, marbofloxacin, and orbifloxacin were determined using minimum inhibitory concentration testing (MICT). Isolates were also tested for sensitivities to enrofloxacin and marbofloxacin using disk-diffusion susceptibility testing (DDST). Isolates were significantly more sensitive to marbofloxacin than to enrofloxacin (z = -4.57; P<0.05) or orbifloxacin (z = -5.02; P<0.05). Agreement was 87% between MICT and DDST for marbofloxacin, with approximately equal numbers of overestimation and underestimation errors. Agreement was 74% between MICT and DDST for enrofloxacin, but DDST tended to overestimate the number of enrofloxacin-susceptible strains. These results suggest that marbofloxacin is more effective against P. aeruginosa than either enrofloxacin or orbifloxacin and that relying on DDST may lead to ineffective enrofloxacin treatment.

  20. Caffeoylquinic acids in leaves of selected Apocynaceae species: Their isolation and content.

    Science.gov (United States)

    Wong, Siu Kuin; Lim, Yau Yan; Ling, Sui Kiong; Chan, Eric Wei Chiang

    2014-01-01

    Three compounds isolated from the methanol (MeOH) leaf extract of Vallaris glabra (Apocynaceae) were those of caffeoylquinic acids (CQAs). This prompted a quantitative analysis of their contents in leaves of V. glabra in comparison with those of five other Apocynaceae species (Alstonia angustiloba, Dyera costulata, Kopsia fruticosa, Nerium oleander, and Plumeria obtusa), including flowers of Lonicera japonica (Japanese honeysuckle), the commercial source of chlorogenic acid (CGA). Compound were isolated by column chromatography, and identified by NMR and MS analyses. CQA content of leaf extracts was determined using reversed-phase HPLC. From the MeOH leaf extract of V. glabra, 3-CQA, 4-CQA, and 5-CQA or CGA were isolated. Content of 5-CQA of V. glabra was two times higher than flowers of L. japonica, while 3-CQA and 4-CQA content was 16 times higher. With much higher CQA content than the commercial source, leaves of V. glabra can serve as a promising alternative source.

  1. Antimicrobial susceptibility survey of pathogens isolated from selected patients in Northern Italy

    Directory of Open Access Journals (Sweden)

    Elisabetta Maioli

    2005-03-01

    Full Text Available The Clinical Microbiology Laboratory of the University of Genoa participated, during the year 2003, in an international antimicrobial surveillance program.The collection of isolates was done according to the site of infection and/or type of patient. Four hundred twenty (420 clinical isolates were analyzed during this year and the frequencies of the different pathogens were investigated. A reference centre carried out susceptibility tests. Oxacillin-resistant Staphylococcus aureus represented 47.6% of all S. aureus isolates from blood stream infections and 33.3% of all S. aureus isolated from skin and soft tissue infections in hospitalised patients.These strains showed resistance to most of the antimicrobial agents evaluated, except vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid which registered 100% of susceptibility. Some isolates from blood stream infections such as E. coli demonstrated resistance to ciprofloxacin (23.3%, levofloxacin (20%, and gatifloxacin (16.6%, and Klebsiella pneumoniae was resistant (18% to all fluoroquinolones tested. Pseudomonas aeruginosa manifested resistance to ciprofloxacin (16.6%, while 27.7% of these strains were resistant both to levofloxacin and gatifloxacin. All the Enterobacter cloacae isolated from blood were susceptible to ciprofloxacin, levofloxacin and gatifloxacin. Haemophilus influenzae and Moraxella catarrhalis collected from community-acquired respiratory tract infections were all inhibited by ciprofloxacin, levofloxacin and gatifloxacin. E. coli isolated from urinary tract infections in hospitalised patients were resistant to ciprofloxacin, levofloxacin and gatifloxacin (2.7%. All Salmonella spp. collected from samples of patients affected by infections of the gastro-intestinal tract were susceptible to all fluoroquinolones. Penicillin resistance in Streptococcus pneumoniae was found in 21.4% of isolates from patients with respiratory tract infections. Fluoroquinolone resistance was

  2. Surface remineralization potential of casein phosphopeptide-amorphous calcium phosphate on enamel eroded by cola-drinks: An in-situ model study

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    Navneet Grewal

    2013-01-01

    Full Text Available Aim: The aim of this study was to investigate the remineralization potential of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP on enamel eroded by cola drinks. Subjects and Methods: A total of 30 healthy subjects were selected from a random sample of 1200 children and divided into two groups of 15 each wherein calcium and phosphorus analyses and scanning electron microscope (SEM analysis was carried out to investigate the remineralization of enamel surface. A total of 30 non-carious premolar teeth were selected from the human tooth bank (HTB to prepare the in-situ appliance. Three enamel slabs were prepared from the same. One enamel slab was used to obtain baseline values and the other two were embedded into the upper palatal appliances prepared on the subjects′ maxillary working model. The subjects wore the appliance after which 30 ml cola drink exposure was given. After 15 days, the slabs were removed and subjected to respective analysis. Statistical Analysis Used: Means of all the readings of soluble calcium and phosphorous levels at baseline,post cola-drink exposure and post cpp-acp application were subjected to statistical analysis SPSS11.5 version.Comparison within groups and between groups was carried out using ANOVA and F-values at 1% level of significance. Results: Decrease in calcium solubility of enamel in the CPP-ACP application group as compared to post-cola drink exposure group (P < 0.05 was seen. Distinctive change in surface topography of enamel in the post-CPP-ACP application group as compared to post-cola drink exposure group was observed. Conclusion: CPP-ACP significantly promoted remineralization of enamel eroded by cola drinks as revealed by significant morphological changes seen in SEM magnification and spectrophotometric analyses.

  3. Evaluation of novel supports for selective and efficient enrichment of phosphorylated peptides

    CSIR Research Space (South Africa)

    Bensadek, D

    2011-06-01

    Full Text Available A series of novel emulsion based supports MagReSynTM TiO2 (A-D) were evaluated using 32P labelled phosphopeptides and nano LC-MS/MS. The performance of MagReSynTM TiO2 (D) was found to be significantly better in terms of selectivity and specificity...

  4. Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains.

    Science.gov (United States)

    Leite, A M O; Miguel, M A L; Peixoto, R S; Ruas-Madiedo, P; Paschoalin, V M F; Mayo, B; Delgado, S

    2015-06-01

    A total of 34 lactic acid bacteria isolates from 4 different Brazilian kefir grains were identified and characterized among a group of 150 isolates, using the ability to tolerate acidic pH and resistance to bile salts as restrictive criteria for probiotic potential. All isolates were identified by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of representative amplicons. Eighteen isolates belonged to the species Leuconostoc mesenteroides, 11 to Lactococcus lactis (of which 8 belonged to subspecies cremoris and 3 to subspecies lactis), and 5 to Lactobacillus paracasei. To exclude replicates, a molecular typing analysis was performed by combining repetitive extragenic palindromic-PCR and random amplification of polymorphic DNA techniques. Considering a threshold of 90% similarity, 32 different strains were considered. All strains showed some antagonistic activity against 4 model food pathogens. In addition, 3 Lc. lactis strains and 1 Lb. paracasei produced bacteriocin-like inhibitory substances against at least 2 indicator organisms. Moreover, 1 Lc. lactis and 2 Lb. paracasei presented good total antioxidative activity. None of these strains showed undesirable enzymatic or hemolytic activities, while proving susceptible or intrinsically resistant to a series of clinically relevant antibiotics. The Lb. paracasei strain MRS59 showed a level of adhesion to human Caco-2 epithelial cells comparable with that observed for Lactobacillus rhamnosus GG. Taken together, these properties allow the MRS59 strain to be considered a promising probiotic candidate. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Isolation and in vitro selection of actinomycetes strains as potential probiotics for aquaculture

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    Milagro García Bernal

    2015-02-01

    Full Text Available Aim: This study was designed to describe a series of in vitro tests that may aid the discovery of probiotic strains from actinomycetes. Materials and Methods: Actinomycetes were isolated from marine sediments using four different isolation media, followed by antimicrobial activity and toxicity assessment by the agar diffusion method and the hemolysis of human blood cells, respectively. Extracellular enzymatic production was monitored by the hydrolysis of proteins, lipids and carbohydrates. Tolerance to different pH values and salt concentrations was also determined, followed by hydrophobicity analysis and genetic identification of the most promising strains. Results: Five out of 31 isolated strains showed antimicrobial activity against three Vibrio species. Three non-hemolytic strains (N7, RL8 and V4 among these active isolates yielded positive results in hydrophobicity tests and exhibited good growth at salt concentrations ranging from 0% to 10%, except strain RL8, which required a salt concentration >0.6%. Although these strains did not grow at pH<3, they showed different enzymatic activities. Phylogenetic analysis revealed that strains N7 and V4 have more than 99% identity with several Streptomyces species, whereas the closest matches to strain RL8 are Streptomyces panacagri and Streptomyces flocculus, with 98% and 98.2% similarity, respectively. Conclusion: Three actinomycetes strains showing probiotic-like properties were discovered using several in vitro tests that can be easily implemented in different institutions around the world.

  6. Virulence potential of Enterococcus gallinarum strains isolated from selected Nigerian traditional fermented foods

    OpenAIRE

    IYABO C. OLADIPO; ABIODUN I. SANNI; SNEHASIKTAS SWARNAKAR

    2014-01-01

    Five Enterococcus isolates from some Nigerian traditional fermented foods were identified as Enterococcus gallinarum by using phenotypic and genotypic tests. Safety properties such as antibiotic susceptibility, virulence gene detection, haemolysin, gelatinase and bacteriocin production were determined using standard methods. There was no resistance to clinically relevant antibiotics. Virulence gene for collagen binding antigen and aggregation substance were detected in 60% of the E. gallinaru...

  7. Selection of high temperature and salinity tolerant Trichoderma isolates with antagonistic activity against Sclerotium rolfsii.

    Science.gov (United States)

    Poosapati, Sowmya; Ravulapalli, Prasad Durga; Tippirishetty, Navaneetha; Vishwanathaswamy, Dinesh Kumar; Chunduri, Sarada

    2014-01-01

    Trichoderma isolates were collected from varied agro-climatic zones of India and screened for high temperature and salinity tolerance. Among all the isolates tested, T. asperellum, TaDOR673 was highly tolerant to heat shock of 52°C with a mean spore count (log c.f.u/ml) of 4.33. The isolate after recovery from heat shock possessed higher germination rate and biomass production compared to its wild counterpart, upon prolonged exposure to 37°C. Under stress, TaDOR673 accumulated >15% of trehalose and >5% of mannose and raffinose compared to the wild type strain signifying their role in stress tolerance. T. asperellum, TaDOR693 and T. asperellum, TaDORS3 were identified as superior salt-tolerant isolates. Interestingly, TaDOR673 also possessed similar tolerance levels to increasing saline concentrations as indicated by its improved colony growth under stress conditions. T. asperellum, TaDOR673 and T. asperellum, TaDOR7316 effectively controlled the collar rot disease in groundnut by 79.7% when screened in vitro and in vivo. Thus, the study identified a potential thermotolerant and saline tolerant strain of Trichoderma, TaDOR673 that could be used as potential bioagent in stressed soils.

  8. Prophages in Enterococcal Isolates from Renal Transplant Recipients: Renal Failure Etiologies Promote Selection of Strains

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    Agnieszka Daca

    2014-01-01

    Full Text Available Infections caused by commensal bacteria may be fatal for the patients under immunosuppressive therapy. This results also from difficulty in identification of high risk strains. Enterococcal infections are increasingly frequent but despite many studies on virulence traits, the difference between commensal and pathogenic strains remains unclear. Prophages are newly described as important elements in competition between strains during colonization, as well as pathogenicity of the strains. Here we evaluate a difference in presence of pp4, pp1, and pp7 prophages and ASA (aggregation substance gene expression in enterococcal isolates from renal transplant recipients (RTx with different etiology of the end-stage renal failure. Prophages sequence was screened by PCR in strains of Enterococcus faecalis isolated from urine and feces of 19 RTx hospitalized at Medical University of Gdansk and 18 healthy volunteers. FLOW-FISH method with use of linear locked nucleic acid (LNA probe was used to assess the ASA gene expression. Additionally, ability of biofilm formation was screened by crystal violet staining method. Presence of prophages was more frequent in fecal isolates from immunocompromised patients than in isolates from healthy volunteers. Additionally, both composition of prophages and ASA gene expression were related to the etiology of renal disease.

  9. Selection of potential probiotic Enterococcus faecium isolated from Portuguese fermented food.

    Science.gov (United States)

    Barbosa, Joana; Borges, Sandra; Teixeira, Paula

    2014-11-17

    Four Enterococcus faecium strains isolated from fermented products were evaluated for potential use as probiotic strains. In addition to efaAfm gene, commonly found in E. faecium food isolates, none of the isolates possessed virulence genes and none had positive reactions for the production of tyramine, histamine, putrescine and cadaverine in the screening medium used. All of these four isolates proved to be resistant to 65 °C. E. faecium 119 did not show antimicrobial activity against any of the target bacteria investigated. E. faecium 85 and 101 inhibited Listeria innocua and E. faecium DSMZ 13590. The strain E. faecium 120 inhibited seven target bacteria (Listeria monocytogenes 7946, L. monocytogenes 7947, L. innocua 2030c, L. innocua NCTC 11286, E. faecium DSMZ 13590, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213) and was chosen as the representative to assess the ability to survive gastrointestinal tract passage simulation, as well as the protective role of two food matrices (skim milk and Alheira) during its passage. For both matrices used, no significant differences (pfaecium strain 120 was shown to be a potential candidate for further investigations as a potential probiotic culture. Copyright © 2014. Published by Elsevier B.V.

  10. Selected technological properties and antibiotic resistance of enterococci isolated from milk

    Directory of Open Access Journals (Sweden)

    Margita Čanigová

    2016-10-01

    Full Text Available The aim of this work was to determine counts of enterococci in raw cow milk, to isolate and identify them, to determinate their antibiotic resistance, ability of lactose fermentation, proteolytic and lipolytic activity in different conditions of cultivation. Counts of enterococci were determined after 48 ±2 h cultivation on Slanetz-Bartley agar at 37 ±1 °C. The counts of enterococci in raw cow milk fluctuated from 1.80 x 102 to 1.77 x 103 CFU.mL-1 with average value 7.25 x 102 CFU.mL-1. Species identifications of enterococci isolates were performed using commercial EN-COCCUS test and confirmed by PCR. Majority of tested isolates (85.7% was included to species E. faecalis. Antibiotic resistance was tested on Mueller-Hinton agar using following antimicrobial discs: vancomycin (VA 30 µg.disc-1, gentamicin (CN 120 µg.disc-1, erythromycin (E 15 µg.disc-1, tetracycline (TE 30 µg.disc-1, ampicillin (AMP 10 µg.disc-1, teicoplanin (TEC 30 µg.disc-1. From 13 isolates of enterococci, 1 strain was resistant to vancomycin, 1 strain to tetracycline and 1 to ampicillin, but higher prevalence of intermediate resistance of isolates was determined to tetracycline (5 strains. Ability of lactose fermentation was monitored by change of titratable acidity in UHT milk after 0, 18, 24, 40 and 48 h of cultivation at temperature 25, 30 and 37 °C. The tested strains of enterococci exhibit low milk acidifying ability. Production of proteolytic enzymes was evaluated after cultivation at temperature 7, 25 and 30 °C after 10 days on nutrient agar no. 2 with sterile skim milk (10% w/v with pH 6.0 and 6.5. Proteolytic activity of tested enterococci strains varied depending on tested temperature and pH. Lipolytic activity was determined similarly like proteolytic activity but on tributyrin agar base with tributyrin (1% w/v. Lipolytic activity of isolated enterococci was very low. The tested strains produced halos with zone in range from 7 to 15 mm regardless of p

  11. Selective loss of chemokine receptor expression on leukocytes after cell isolation.

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    Juan C Nieto

    Full Text Available Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB plus erythrocyte lysis and density gradient isolation (Ficoll. Most WB monocytes were CCR2+ (93.5 ± 2.9% whereas 32.8 ± 6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001. Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4 ± 7.5% and 57.1 ± 5.5%; Ficoll: 29.5 ± 2.2% and 5.4 ± 4.3% respectively (p<0.01. Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5 ± 0.4 and Ficoll: MFI 3.3 ± 0.1 (p<0.05. Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2 ± 4.5% and Ficoll: 55 ± 4.1% (p<0.01. The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.

  12. Phylodynamic analysis of clinical and environmental Vibrio cholerae isolates from Haiti reveals diversification driven by positive selection.

    Science.gov (United States)

    Azarian, Taj; Ali, Afsar; Johnson, Judith A; Mohr, David; Prosperi, Mattia; Veras, Nazle M; Jubair, Mohammed; Strickland, Samantha L; Rashid, Mohammad H; Alam, Meer T; Weppelmann, Thomas A; Katz, Lee S; Tarr, Cheryl L; Colwell, Rita R; Morris, J Glenn; Salemi, Marco

    2014-12-23

    Phylodynamic analysis of genome-wide single-nucleotide polymorphism (SNP) data is a powerful tool to investigate underlying evolutionary processes of bacterial epidemics. The method was applied to investigate a collection of 65 clinical and environmental isolates of Vibrio cholerae from Haiti collected between 2010 and 2012. Characterization of isolates recovered from environmental samples identified a total of four toxigenic V. cholerae O1 isolates, four non-O1/O139 isolates, and a novel nontoxigenic V. cholerae O1 isolate with the classical tcpA gene. Phylogenies of strains were inferred from genome-wide SNPs using coalescent-based demographic models within a Bayesian framework. A close phylogenetic relationship between clinical and environmental toxigenic V. cholerae O1 strains was observed. As cholera spread throughout Haiti between October 2010 and August 2012, the population size initially increased and then fluctuated over time. Selection analysis along internal branches of the phylogeny showed a steady accumulation of synonymous substitutions and a progressive increase of nonsynonymous substitutions over time, suggesting diversification likely was driven by positive selection. Short-term accumulation of nonsynonymous substitutions driven by selection may have significant implications for virulence, transmission dynamics, and even vaccine efficacy. Cholera, a dehydrating diarrheal disease caused by toxigenic strains of the bacterium Vibrio cholerae, emerged in 2010 in Haiti, a country where there were no available records on cholera over the past 100 years. While devastating in terms of morbidity and mortality, the outbreak provided a unique opportunity to study the evolutionary dynamics of V. cholerae and its environmental presence. The present study expands on previous work and provides an in-depth phylodynamic analysis inferred from genome-wide single nucleotide polymorphisms of clinical and environmental strains from dispersed geographic settings in

  13. A Multicenter Study: North American Islet Donor Score in Donor Pancreas Selection for Human Islet Isolation for Transplantation.

    Science.gov (United States)

    Wang, Ling-Jia; Kin, Tatsuya; O'Gorman, Doug; Shapiro, A M James; Naziruddin, Bashoo; Takita, Morihito; Levy, Marlon F; Posselt, Andrew M; Szot, Gregory L; Savari, Omid; Barbaro, Barbara; McGarrigle, James; Yeh, Chun Chieh; Oberholzer, Jose; Lei, Ji; Chen, Tao; Lian, Moh; Markmann, James F; Alvarez, Alejandro; Linetsky, Elina; Ricordi, Camillo; Balamurugan, A N; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Hering, Bernhard J; Bottino, Rita; Trucco, Massimo; Liu, Chengyang; Min, Zaw; Li, Yanjing; Naji, Ali; Fernandez, Luis A; Ziemelis, Martynas; Danobeitia, Juan S; Millis, J Michael; Witkowski, Piotr

    2016-01-01

    Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70-0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality.

  14. Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Sang-Moon; Moulaei, Tinoush; Lim, Dan; Bang, Jeong K.; Park, Jung-Eun; Shenoy, Shilpa R.; Liu, Fa; Kang, Young H.; Liao, Chenzhong; Soung, Nak-Kyun; Lee, Sunhee; Yoon, Do-Young; Lim, Yoongho; Lee, Dong-Hee; Otaka, Akira; Appella, Ettore; McMahon, James B.; Nicklaus, Marc C.; Burke, Jr., Terrence R.; Yaffe, Michael B.; Wlodawer, Alexander; Lee, Kyung S.; (MIT); (Konkuk); (Korea Basic Science Institute); (SAIC); (NCI); (University of Tokushima)

    2009-09-14

    Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

  15. [Selection of isolates of entomopathogenic fungus Beauveria bassiana (Ascomycetes: Clavicipitaceae) for control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)].

    Science.gov (United States)

    Barci, Leila A G; de Almeida, José Eduardo M; de Campos Nogueira, Adriana H; Zappelini, Luciano O; do Prado, Angelo P

    2009-12-01

    This study was carried out to select isolates of the entomopathogenic fungus Beauveria bassiana with pathogenic potential to control the Rhipicephalus (Boophilus) microplus tick. The effectiveness of thirty isolates was first tested at a concentration of 5 x 108 conidia.mL(-1). Of these, eight were evaluated (IBCB01, IBCB02, IBCB07, IBCB17, IBCB21, IBCB74, IBCB149, IBCB165) and showed an effectiveness between 90 and 99%; thirteen (IBCB03, IBCB14, IBCB16, IBCB24, IBCB95, IBCB97, IBCB102, IBCB141, IBCB146, IBCB147, IBCB150, IBCB154, IBCB157) between 80 and 89,5%; six (IBCB47, IBCB75, IBCB84, IBCB145, IBCB161, IBCB164) between 70 and 79%, and only two (IBCB13 and IBCB143) had lower pathogenicity (70% or below). In the second step of the study, the five more effective strains in the first phase of the experiment (IBCB01, IBCB07, IBCB21, IBCB66, IBCB165) were analyzed comparatively. Based on in vitro results, it can be concluded that IBCB66 and IBCB21 are the isolates with higher potential for field control of R. (B.) microplus. IBCB01, IBCB07, IBCB21, IBCB66 e IBCB165 isolates were submitted to a conidial production test using a rice-based substrate. The best mass production of the entomopathogenic fungus was obtained with the IBCB66 strain.

  16. Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus

    Directory of Open Access Journals (Sweden)

    Derbel Maher

    2001-12-01

    Full Text Available Abstract Background Scorpion venom contains insect and mammal selective toxins. We investigated the venom of the South Indian red scorpion, Mesobuthus tamulus for the purpose of identifying potent insecticidal peptide toxins. Results A lepidopteran-selective toxin (Buthus tamulus insect toxin; ButaIT has been isolated from this venom. The primary structure analysis reveals that it is a single polypeptide composed of 37 amino acids cross-linked by four disulfide bridges with high sequence homology to other short toxins such as Peptide I, neurotoxin P2, Lqh-8/6, chlorotoxin, insectotoxin I5A, insect toxin 15 and insectotoxin I1. Three dimensional modeling using Swiss automated protein modeling server reveals that this toxin contains a short α-helix and three antiparallel β-strands, similar to other short scorpion toxins. This toxin is selectively active on Heliothis virescens causing flaccid paralysis but was non-toxic to blowfly larvae and mice. Conclusion This is the first report of a Heliothine selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.

  17. Photostick: a method for selective isolation of target cells from culture.

    Science.gov (United States)

    Chien, Miao-Ping; Werley, Christopher A; Farhi, Samouil L; Cohen, Adam E

    2015-03-01

    Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The "photostick" technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns.

  18. Development of a Selective Culture Medium for Primary Isolation of the Main Brucella Species▿

    OpenAIRE

    Miguel, María Jesús de; Marín, Clara M.; Muñoz, Pilar M.; Dieste, L.; Grilló, María Jesús; Blasco, José M

    2011-01-01

    Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis ...

  19. The Patterns Of Selected Antibiotics Sensitivity And Resistance To Staphylococcus Aureus Isolates

    Directory of Open Access Journals (Sweden)

    Stanslaus Kiilu Musyoki

    2015-08-01

    Full Text Available Complications of Staphylococcus aureus infection have greatly increased in recent past because of the many invasive procedures increased cases of immunocompromised individuals and the uprising trends in increased antimicrobial resistance of S. aureus strains. Despite of these available information and by contrast with developed countries S. aureus associated disease are ranked low on the public-health agenda in Kenya and other developing countries. Therefore there is due reason to undertake an investigation and report the trends and patterns in a thorough manner majorly and especially regarding the antimicrobial resistance. The aim of this study was thus to determine the levels of drug resistance of Staphylococcus aureus to various classes of antibiotics. This data is of significance in improving baseline data on antibiotic resistance of S. aureus isolated from human clinical specimens for the prudent use of antibiotics and the coming up with policies on control programs. All culture isolates were confirmed as Staphylococcus aureus genus by various tests That is gram staining catalase and oxidase. Catalase positive gram positive and oxidase negative isolates were defined as Staphylococcus. Further analyses by mannitol salt agar fermentation of the isolates and positive coagulase tests indicated Staphylococcus aureus. The area of clearance of sensitivity and tolerance was measured in millimeters and categorized as sensitive resistant or intermediate. The present study reported that S. aureus was most sensitive to Azithromycin whereby 46 61 samples were sensitive. Penicillin on the hand was least sensitive showing 29 level of sensitivity. Methicillin Gentamicin had more than 50 level of sensitivity That is 41 55 and 40 53 respectively. Other antibiotic drugs including ampicillin augmentin and tetracycline demonstrated less than 50 sensitivity That is 29 39 32 43 and 33 44 respectively. Drug resistance for S. aureus was therefore reported to be

  20. SELECTED PROPERTIES OF LACTIC ACID BACTERIA ISOLATED FROM RAW COW'S MILK

    Directory of Open Access Journals (Sweden)

    Miroslav Kročko

    2012-02-01

    Full Text Available Normal 0 21 false false false MicrosoftInternetExplorer4 For the identification of lactic acid bacteria derived from raw cow's milk, 151 colonies were isolated. The grow conditions of lactic acid bacteria were at temperature 37 °C for 3 days on MRS medium. Based on microscopical preparation, negative catalase and Gram-positive test were 81 isolates confirmed as genus Lactobacillus. Out of these, 9 isolates were evaluated for acidifying activity in UHT milk at 25 °C, 30 °C and 37 °C at regular intervals during 24 hours. The average count of NSLAB lactobacilli in raw cow's milk reached the value 1.54.104 KTJ.ml-1. It was found that all tested strains of lactobacilli did not cause significant changes of titratable acidity in milk at 25 °C and 30 °C. Only one strain significantly improved the titratable acidity of milk at 37 °C after 24 hours. The acidity reached the value from 7.5 °SH to 41.9 °SH. This strain was confirmed by PCR method as Lactobacillus helveticus.doi:10.5219/177

  1. Reactive landing of gas-phase ions as a tool for the fabrication of metal oxide surfaces for in situ phosphopeptide enrichment.

    Science.gov (United States)

    Blacken, Grady R; Volný, Michael; Diener, Matthew; Jackson, Karl E; Ranjitkar, Pratistha; Maly, Dustin J; Turecek, Frantisek

    2009-06-01

    Zirconium, titanium, and hafnium oxide-coated stainless steel surfaces are fabricated by reactive landing of gas-phase ions produced by electrospray ionization of group IVB metal alkoxides. The surfaces are used for in situ enrichment of phosphopeptides before analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To evaluate this method we characterized ZrO(2) (zirconia) surfaces by (1) comparison with the other group IVB metal oxides of TiO(2) (titania) and HfO(2) (hafnia), (2) morphological characterization by SEM image analysis, and (3) dependence of phosphopeptide enrichment on the metal oxide layer thickness. Furthermore, we evaluated the necessity of the reactive landing process for the construction of useful metal oxide surfaces by preparing surfaces by electrospray deposition of Zr, Ti, and Hf alkoxides directly onto polished metal surfaces at atmospheric pressure. Although all three metal oxide surfaces evaluated were capable of phosphopeptide enrichment from complex peptide mixtures, zirconia performed better than hafnia or titania as a result of morphological characteristics illustrated by the SEM analysis. Metal oxide coatings that were fabricated by atmospheric pressure deposition were still capable of in situ phosphopeptide enrichment, although with inferior efficiency and surface durability. We show that zirconia surfaces prepared by reactive landing of gas-phase ions can be a useful tool for high throughput screening of novel phosphorylation sites and quantitation of phosphorylation kinetics.

  2. Effects of casein phosphopeptide amorphous calcium fluoride phosphate paste on white spot lesions and dental plaque after orthodontic treatment: a 3-month follow-up

    NARCIS (Netherlands)

    Beerens, M.W.; van der Veen, M.H.; van Beek, H.; ten Cate, J.M.

    2010-01-01

    The effects of casein phosphopeptide amorphous calcium fluoride phosphate (CPP-ACFP) paste vs. control paste on the remineralization of white spot caries lesions and on plaque composition were tested in a double-blind prospective randomized clinical trial. Fifty-four orthodontic patients, with

  3. In vitro screening of selected probiotic properties of Lactobacillus strains isolated from traditional fermented cabbage and cucumber.

    Science.gov (United States)

    Zielińska, Dorota; Rzepkowska, Anna; Radawska, Anna; Zieliński, Konrad

    2015-02-01

    Most important during probiotic selection are gastric acid and bile tolerance, the adhesion to the luminal epithelium to colonize the lower gastrointestinal tract of a human and safety for human consumption. The aim of this study was to evaluate the selected probiotic in vitro properties of Lactobacillus spp. Strains isolated from traditional fermented food. A total 38 strains were isolated from the pickled samples and 14 were identified as Lactobacillus spp. The survival of almost all strains after incubation at pH 2.5 did not change markedly, and remained at above 90 % (10(9) CFU/mL). The strains also exhibited a high survival rate at pH 3.5 (>90 %), whereas pH 1.5 all were died. Just four strains could survive 90 min. at pH 1.5 (Safety evaluation excluded four of tested strains as candidate probiotics, according to antibiotic resistance patterns and certain metabolic activities. On the basis on the results 10 of the selected Lactobacillus strains are safe and can survive under gastrointestinal conditions, which requires them to future in vitro and in vivo probiotic studies.

  4. Isolation of Cu Atoms in Pd Lattice: Forming Highly Selective Sites for Photocatalytic Conversion of CO2 to CH4.

    Science.gov (United States)

    Long, Ran; Li, Yu; Liu, Yan; Chen, Shuangming; Zheng, Xusheng; Gao, Chao; He, Chaohua; Chen, Nanshan; Qi, Zeming; Song, Li; Jiang, Jun; Zhu, Junfa; Xiong, Yujie

    2017-03-29

    Photocatalytic conversion of CO2 to CH4, a carbon-neutral fuel, represents an appealing approach to remedy the current energy and environmental crisis; however, it suffers from the large production of CO and H2 by side reactions. The design of catalytic sites for CO2 adsorption and activation holds the key to address this grand challenge. In this Article, we develop highly selective sites for photocatalytic conversion of CO2 to CH4 by isolating Cu atoms in Pd lattice. According to our synchrotron-radiation characterizations and theoretical simulations, the isolation of Cu atoms in Pd lattice can play dual roles in the enhancement of CO2-to-CH4 conversion: (1) providing the paired Cu-Pd sites for the enhanced CO2 adsorption and the suppressed H2 evolution; and (2) elevating the d-band center of Cu sites for the improved CO2 activation. As a result, the Pd7Cu1-TiO2 photocatalyst achieves the high selectivity of 96% for CH4 production with a rate of 19.6 μmol gcat-1 h-1. This work provides fresh insights into the catalytic site design for selective photocatalytic CO2 conversion, and highlights the importance of catalyst lattice engineering at atomic precision to catalytic performance.

  5. Selective Isolation of Trypsin Inhibitor and Lectin from Soybean Whey by Chitosan/Tripolyphosphate/Genipin Co-Crosslinked Beads

    Directory of Open Access Journals (Sweden)

    Yu-Lung Chang

    2014-06-01

    Full Text Available Selective isolation of Kunitz trypsin inhibitor (KTI and lectin from soybean whey solutions by different types of chitosan beads was investigated. The chitosan beads were co-crosslinked with tripolyphosphate/genipin in solutions at pH 5, 7 or 9 (CB5, CB7, CB9. The maximum adsorption ratios of chitosan beads to KTI and lectin were observed at pH 4.4 and 5.4, respectively; highly selective separation was also demonstrated at these pHs. The adsorption ratios increased with temperature, rising between 5 and 25 °C. CB9 produced the best adsorption ratio, followed by CB7 then CB5. The critical interaction governing absorption of chitosan beads to KTI and lectin could be hydrogen bonding. At pH 9, KTI and lectin desorbed efficiently from CB7 with desorption ratios of 80.9% and 81.4%, respectively. The desorption was most likely caused predominantly by electrostatic repulsion. KTI and lectin can effectively be selectively isolated from soybean whey using this novel separation technique.

  6. Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

    Science.gov (United States)

    Yang, Xiaojian; Brisbin, Jennifer; Yu, Hai; Wang, Qi; Yin, Fugui; Zhang, Yonggang; Sabour, Parviz; Sharif, Shayan; Gong, Joshua

    2014-01-01

    Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3-1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7) CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (10(4) CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one of the strategies for controlling Salmonella infection in chickens.

  7. In vitro evaluation of casein phosphopeptide-amorphous calcium phosphate effect on the shear bond strength of dental adhesives to enamel

    Directory of Open Access Journals (Sweden)

    Niloofar Shadman

    2015-01-01

    Full Text Available Background: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP is applied for remineralization of early caries lesions or tooth sensitivity conditions and may affect subsequent resin bonding. This in vitro study investigated the effect of CPP-ACP on the shear bond strength of dental adhesives to enamel. Materials and Methods: Sixty extracted human molar teeth were selected and randomly divided into three groups and six subgroups. Buccal or lingual surfaces of teeth were prepared to create a flat enamel surface. Adhesives used were Tetric N-Bond, AdheSE and AdheSE One F. In three subgroups, before applying adhesives, enamel surfaces were treated with Tooth Mousse CPP-ACP for one hour, rinsed and stored in 37°C temperature with 100% humidity. This procedure was repeated for 5 days and then adhesives were applied and Tetric N-Ceram composite was adhered to the enamel. This procedure was also fulfilled for the other three subgroups without CPP-ACP treatment. After 24 hour water storage, samples were tested for shear bond strength test in a universal testing machine. Failure modes were determined by stereomicroscope. Data were analyzed by t-test and one-way analysis of variance with P 0.05. In non-applied CPP-ACP subgroups, there were statistically significant differences among all subgroups. Tetric N-Bond had the highest and AdheSE One F had the lowest shear bond strength. Conclusion: CPP-ACP application reduces the shear bond strength of AdheSE and AdheSE One F to enamel but not Tetric N-Bond.

  8. Identification of Highly Potent and Selective α-Glucosidase Inhibitors with Antiglycation Potential, Isolated from Rhododendron arboreum

    OpenAIRE

    Rabia Raza; Zaitoon Ilyas; Sajid Ali; Muhammad Nisar; Muhammad Younas Khokhar; Jamshed Iqbal

    2015-01-01

    This study explored antidiabetic potential of eight known pure compounds, isolated from the bark of Rhododendron arboreum. Invitro studies of these compounds against α and β-glucosidases revealed them as very potent and selective inhibitors of α-glucosidase. Compound 7 (3-O-acetylursolic acid) was found to be the most potent inhibitor of α-glucosidase with 3.3±0.1µM IC 50 value which was many folds higher than standard inhibitor acarbose. Antiglycation studies of compounds showed that all com...

  9. Temporally isolated lineages of Pink salmon reveal unique signatures of selection on distinct pools of standing genetic variation

    DEFF Research Database (Denmark)

    Limborg, Morten; Waples, R.K.; Seeb, J.E.

    2014-01-01

    gorbuscha) using genome scans coupled with inference from a haploid-assisted linkage map. Pink salmon have a strict 2-year semelparous life history which has resulted in temporally isolated (allochronic) lineages that remain sympatric through sharing of spawning habitats in alternate years. The lineages...... differ in a range of adaptive traits, suggesting different genetic backgrounds. We used genotyping by sequencing of haploids to generate a high-density linkage map with 7035 loci and screened an existing panel of 8036 loci for signatures of selection. The linkage map enabled identification of novel...

  10. Isolation, Characterization, and Selection of Molds Associated to Fermented Black Table Olives

    DEFF Research Database (Denmark)

    Bavaro, Simona L.; Susca, Antonia; Frisvad, Jens Christian

    2017-01-01

    Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food...... safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly...

  11. Selective-Differential Medium for Isolation and Differentiation of Pectinatus from Other Brewery Microorganisms

    OpenAIRE

    Lee, Sun Y.; Moore, Sharon E.; Mabee, Michael S.

    1981-01-01

    An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and ...

  12. Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kouprina, Natalay; Larionov, Vladimir

    2008-01-01

    Here, we describe a protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC). The method is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae between genomic DNA and a linearized TAR cloning vector containing targeting sequences homologous to a region of interest. Recombination between the vector and homologous sequences in the co-transformed mammalian DNA results in the establishment of a YAC that is able to propagate, segregate and be selected for in yeast. Yield of gene-positive clones varies from 1% to 5%. The entire procedure takes 2 weeks to complete once the TAR vector is constructed and genomic DNA is prepared. The TAR cloning method has a broad application in functional and comparative genomics, long-range haplotyping and characterization of chromosomal rearrangements, including copy number variations.

  13. Selection and characterisation of an HD1-like Bacillus thuringiensis isolate with a high insecticidal activity against Spodoptera littoralis (Lepidoptera: Noctuidae).

    Science.gov (United States)

    Azzouz, Hichem; Kebaili-Ghribi, Jihene; ben Farhat-Touzri, Dalel; Daoud, Fatma; Fakhfakh, Ines; Tounsi, Slim; Jaoua, Samir

    2014-08-01

    Spodoptera littoralis (Boisduval) larvae are known by their susceptibility to Bacillus thuringiensis subsp. aizawai strains. In order to prevent the appearance of B. thuringiensis (Bt) resistance and to develop economical Bt-based biopesticides, the selection and the characterisation of a B. thuringiensis isolate toxic against S. littoralis larvae and overproducing δ-endotoxins were investigated. Among 124 Tunisian B. thuringiensis isolates assessed against S. littoralis larvae, four isolates showed toxicity similar to and higher than the toxicity of the aizawai strain HD133 and the kurstaki strain HD1 respectively. The plasmid pattern of the selected isolates was similar to that of HD1. Polymerase chain reaction (PCR) analysis using specific primers revealed that these isolates present different gene contents. The only detected gene encoding Spodoptera-specific toxin was cry9. The selected isolates were found to produce bipyramidal and cubic crystals. The assessment of δ-endotoxin production by these isolates showed that BUPM28 produced 43.71 and 80.81% more δ-endotoxin than HD1 and HD133 respectively. The application of osmotic or heat shock stress on the BUPM28 isolate made it possible to enhance δ-endotoxin production by 22 and 23% respectively. On the basis of its potent insecticidal activity and its high level of δ-endotoxin production, the BUPM28 isolate can be considered to be an effective alternative for the control of S. littoralis. © 2013 Society of Chemical Industry.

  14. Selection of lactic acid bacteria isolated from Tunisian cereals and exploitation of the use as starters for sourdough fermentation.

    Science.gov (United States)

    Mamhoud, Asma; Nionelli, Luana; Bouzaine, Taroub; Hamdi, Moktar; Gobbetti, Marco; Rizzello, Carlo Giuseppe

    2016-05-16

    Wheat bread is the most popular staple food consumed in Tunisia and, despite the niche production of some typical breads (e.g. Tabouna, Mlawi, Mtabga), the major part is currently produced with baker's yeast at industrial or, mainly, at artisanal level, while the use of sourdough fermentation is rarely reported. Considering the growing national demand for cereal baked goods, it can be hypothesized that sourdough fermentation through the use of selected lactic acid bacteria as starters could improve the overall quality and the diversification of local products. Different cereal grains were collected from the regions of Ariana, Bizerta, Beja Nabeul, and Seliana, and the autochthonous lactic acid bacteria were isolated, identified, characterized and selected on the basis of the kinetics of acidification, the proteolytic activity, and the quotient of fermentation. Lactobacillus curvatus MA2, Pediococcus pentosaceus OA2, and Pediococcus acidilactici O1A1 were used together as mixed starter to obtain a selected sourdough. According to the backslopping procedure, a type I sourdough was made from a Tunisian flour (spontaneous sourdough). Compared to the use of the spontaneous sourdough, the one obtained with selected and mixed starters by a unique fermentation step, favored the increase of the concentrations of organic acids, phenols, and total free amino acids, the most suitable quotient of fermentation, and the most intense phytase and antioxidant activities, that increased ca. 20% compared to the control. Moreover, the selected starters improved the in vitro protein digestibility (ca. 82% when selected sourdough was used), textural and sensory features of the breads, as determined by textural profile analysis and panel test, respectively. This study aimed at exploiting the potential of selected autochthonous lactic acid bacteria and extending the use of a sourdough (type II), thanks to the set-up of a two-step fermentation protocol designed for application at the

  15. Prevalence and Bacterial Isolates of Mastitis in Dairy Farms in Selected Districts of Eastern Harrarghe Zone, Eastern Ethiopia

    Directory of Open Access Journals (Sweden)

    Tesfaheywet Zeryehun

    2017-01-01

    Full Text Available The study was conducted from November 2015 to April 2016 to estimate the prevalence of clinical and subclinical mastitis in lactating cows, to assess the associated risk factors, and to isolate the major bacterial pathogens in dairy farms in selected district of Eastern Harrarghe Zone, Eastern Ethiopia. The study was carried out in 384 dairy cows based on data collection, farm visit, animal examination, California mastitis test (CMT, and isolation bacterial pathogens using standard techniques. In the present study the overall mastitis at cow level was 247 (64.3%. The prevalence of clinical and subclinical mastitis and quarter level prevalence for clinical and subclinical mastitis were 12.5% and 51.8% at cow level and 10.7% and 46.4% at quarter level, respectively. Clinically, 101 (6.6% quarters which belong to 75 (19.5% animals were found to be with blind teat. In the present study prevalence of mastitis was significantly associated with parity and age (p<0.05. Bacteriological examination of milk sample revealed 187 isolates where coagulase negative Staphylococcus species (CNS (34.2% was the predominant species while Streptococcus faecalis (2.1% was identified as the least bacteria. The present study concluded that prevalence of mastitis particularly the subclinical mastitis was major problem of dairy cows in the area and hence warrants serious attention.

  16. Fully automated synthesis of (phosphopeptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization

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    Goldstein David J

    2005-01-01

    Full Text Available Abstract Background Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src. Results The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR autophosphorylation site (pY1173 antibody ELISA (9H2 was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phosphopeptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of

  17. Selection and geographic isolation influence hummingbird speciation: genetic, acoustic and morphological divergence in the wedge-tailed sabrewing (Campylopterus curvipennis

    Directory of Open Access Journals (Sweden)

    Ornelas Juan

    2011-02-01

    Full Text Available Abstract Background Mesoamerica is one of the most threatened biodiversity hotspots in the world, yet we are far from understanding the geologic history and the processes driving population divergence and speciation for most endemic taxa. In species with highly differentiated populations selective and/or neutral factors can induce rapid changes to traits involved in mate choice, promoting reproductive isolation between allopatric populations that can eventually lead to speciation. We present the results of genetic differentiation, and explore drift and selection effects in promoting acoustic and morphological divergence among populations of Campylopterus curvipennis, a lekking hummingbird with an extraordinary vocal variability across Mesoamerica. Results Analyses of two mitochondrial genes and ten microsatellite loci genotyped for 160 individuals revealed the presence of three lineages with no contemporary gene flow: C. c. curvipennis, C. c. excellens, and C. c. pampa disjunctly distributed in the Sierra Madre Oriental, the Tuxtlas region and the Yucatan Peninsula, respectively. Sequence mtDNA and microsatellite data were congruent with two diversification events: an old vicariance event at the Isthmus of Tehuantepec (c. 1.4 Ma, and a more recent Pleistocene split, isolating populations in the Tuxtlas region. Hummingbirds of the excellens group were larger, and those of the pampa group had shorter bills, and lineages that have been isolated the longest shared fewer syllables and differed in spectral and temporal traits of a shared syllable. Coalescent simulations showed that fixation of song types has occurred faster than expected under neutrality but the null hypothesis that morphological divergence resulted from drift was not rejected. Conclusions Our phylogeographic analyses uncovered the presence of three Mesoamerican wedge-tailed sabrewing lineages, which diverged at different time scales. These results highlight the importance of the

  18. Selection of Lactic Acid Bacteria with Probiotic Potential Isolated from the Fermentation Process of "Cupuaçu" (Theobroma grandiflorum).

    Science.gov (United States)

    Ornellas, Roberta Maria Santos; Santos, Tiza Teles; Arcucio, Leonardo Borges; Sandes, Sávio Henrique Cicco; Oliveira, Mayara Messias; Dias, Cristiano Villela; de Carvalho Silva, Samuel; Uetanabaro, Ana Paula Trovatti; Vinderola, Gabriel; Nicoli, Jacques Robert

    2017-01-01

    In the present study, nine lactic acid bacteria isolated from the fermentation process of "cupuaçu" (Theobroma grandiflorum) were selected for probiotic use. In vitro (resistance to gastrointestinal environment, in vitro antagonism and co-aggregation with pathogens) and in vivo (intestinal colonization and ex vivo antagonism in germ-free mice, cumulative mortality, translocation to liver and spleen, histopathological examination of liver and ileum and mRNA cytokine gene expression during an experimental infection with S. Typhimurium) assays were used. Among the nine Lactobacillus strains isolated from the "cupuaçu" fermentation, L. plantarum 81 and L. plantarum 90 were selected as potential probiotics based on better results obtained in in vitro evaluations (production of diffusible inhibitory compounds and co-aggregation) as well as in vivo experiments (resistance to gastrointestinal environment, ex vivo antagonism, higher survival after enteropathogen challenge, lower hepatic translocation of enteropathogen, lower histopathological lesions in ileum and liver and anti-inflammatory pattern of immunological response). Concluding, L. plantarum 81 and L. plantarum 90 showed in vitro and in vivo capacities for probiotic use through different mechanisms of protection and its origin would allow an easier adaptation in an alimentary matrix for its administration.

  19. Isolation, identification and selection of antagonistic yeast against Alternaria alternata infection and tenuazonic acid production in wine grapes from Argentina.

    Science.gov (United States)

    Prendes, Luciana P; Merín, María G; Fontana, Ariel R; Bottini, Rubén A; Ramirez, María L; Morata de Ambrosini, Vilma I

    2017-11-08

    Epiphytic isolates with yeast characteristics from grapes of the Malbec cultivar were obtained in order to find antagonists against Alternaria alternata. From a total of 111 isolates, 82% corresponded to the yeast-like organism Aureobasidium pullulans and the rest to the non-Saccharomyces yeasts Hanseniaspora uvarum (6.3%), Metschnikowia pulcherrima or spp. (5.4%), Cryptoccocus laurentti II (2.7%), Starmerella bacilaris or Candida zemplinina (2.7%) and Rhodotorula spp. (0.9%). The 22.4% (15 out of 67) of epiphytic yeasts and yeast-like organisms evaluated were able to reduce A. alternata infection from 0.0 to 4.4% when applied 2h previous to pathogen inoculation on wounds of grape berries. From these selected strains, 14 out of 15 strains completely prevented A. alternata infection (0.0%), which implies potential for field application. All Metschnikowia (pulcherrima or spp.), S. bacillaris and almost all H. uvarum evaluated strains showed antagonist capability against A. alternata. Meanwhile, none of the lesser nutritional requirement strains belonging to A. pullulans, Cr. laurenti II and Rhodotorula spp. did. All the yeasts with capacity to prevent A. alternata infection also reduced tenuazonic acid (TA) production by 81.2 to 99.8%, finding TA levels similar to negative controls. Therefore, the epiphytic yeasts selected are promising as biological control agents against Alternaria infection and toxin production in grapes for winemaking. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Parallel signatures of selection in temporally isolated lineages of pink salmon

    DEFF Research Database (Denmark)

    Seeb, L. W.; Waples, R. K.; Limborg, M. T.

    2014-01-01

    Studying the effect of similar environments on diverse genetic backgrounds has long been a goal of evolutionary biologists with studies typically relying on experimental approaches. Pink salmon, a highly abundant and widely ranging salmonid, provide a naturally occurring opportunity to study the ...... be particularly informative in understanding adaptive evolution in pink salmon and exploring how differing genetic backgrounds within a species respond to selection from the same natural environment......Studying the effect of similar environments on diverse genetic backgrounds has long been a goal of evolutionary biologists with studies typically relying on experimental approaches. Pink salmon, a highly abundant and widely ranging salmonid, provide a naturally occurring opportunity to study...... in the southern pair from Puget Sound than in the northern Alaskan population pairs. We identified 15 SNPs reflecting signatures of parallel selection using both a differentiation-based method (BAYESCAN) and an environmental correlation method (BAYENV). These SNPs represent genomic regions that may...

  1. Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells.

    Science.gov (United States)

    Henry, Michael; Coleman, Orla; Prashant; Clynes, Martin; Meleady, Paula

    2017-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is one of the most important post-translational modifications that regulates many biological processes. The phosphoproteome has not been studied in any great detail in recombinant Chinese hamster ovary (CHO) cells to date despite phosphorylation playing a crucial role in regulating many molecular and cellular processes relevant to bioprocess phenotypes including, for example, transcription, translation, growth, apoptosis, and signal transduction. In this chapter, we provide a protocol for the phosphoproteomic analysis of Chinese hamster ovary cells using phosphopeptide enrichment with metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) techniques, followed by site-specific identification of phosphorylated residues using LC-MS (MS2 and MS3) strategies.

  2. Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry

    DEFF Research Database (Denmark)

    Bennett, Keiryn L; Stensballe, Allan; Podtelejnikov, Alexandre V

    2002-01-01

    A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed...... ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide...... the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium...

  3. Growth inhibition by nitrocompounds of selected uric acid-utilizing microorganisms isolated from poultry manure.

    Science.gov (United States)

    Kim, W K; Anderson, R C; Ratliff, A L; Nisbet, D J; Ricke, S C

    2006-01-01

    The overall objective of this study was to evaluate the potential ability of nitrocompounds to reduce ammonia volatilization by inhibiting uric acid-utilizing microorganisms. Experiment I was conducted to evaluate the effects of nitrocompounds on the growth of uric acid-utilizing microorganisms isolated from poultry manure during six-hour incubation. There were five treatments: (1) control, (2) 50 mM nitroethane, (3) 50 mM nitroethanol, (4) 50 mM nitropropanol, and (5) 50 mM nitropropionic acid. Optical density values of nitrocompounds were significantly lower than that of control at two, four, and six hours. Plate counts of uric acid-utilizing microorganisms after six-hour incubation exhibited that nitrocompounds greatly reduced the growth of these microorganisms except for the nitroethane (P growth of uric acid-utilizing microorganisms compared to non-nitrocompounds such as ethanol, propanol, and propionic acid. Experiments II and III consisted of seven treatments: (1) control, (2) nitroethanol, (3) nitropropanol, (4) nitropropionic acid, (5) ethanol, (6) propanol, and (7) propionic acid. The incubation times of Experiments II and III were 6 and 24 h, respectively. The nitrocompounds were significantly more successful in inhibiting growth of uric acid-utilizing microorganisms compared to those non-nitrocompounds. These results suggest that nitrocompounds exhibit potential to reduce ammonia volatilization in poultry manure by inhibiting growth of uric acid-utilizing microorganisms.

  4. Antimicrobial activity, toxicity and selectivity index of two biflavonoids and a flavone isolated from Podocarpus henkelii (Podocarpaceae) leaves.

    Science.gov (United States)

    Bagla, Victor P; McGaw, Lyndy J; Elgorashi, Esam E; Eloff, Jacobus N

    2014-10-08

    Different parts of Podocarpus henkelii have been used in many cultures around the world to treat ailments such as cholera, stomach diseases, rheumatism, cancer, canine distemper in dogs and gall sickness in cattle. The aim of this study was to evaluate the biological activity and toxicity of isolated compounds from Podocarpus henkelii after an earlier study indicated a promising activity in crude extracts against viral pathogens of veterinary importance. The antibacterial and antifungal activity of two biflavonoids 7, 4', 7", 4"'-tetramethoxy amentoflavone (TMA), isoginkgetin (IGG) and podocarpus flavone-A (PFA) isolated from the leaves of Podocarpus henkelii were determined using a serial microplate dilution method with tetrazolium violet as growth indicator. The cytotoxicity of compounds TMA and IGG were determined on different cell types using a tetrazolium-based colorimetric cellular assay (MTT). The Ames test was used to determine their mutagenic activities. TMA had reasonable antifungal activity against Aspergillus fumigatus (MIC = 30 μg/ml). IGG had a wide spectrum of activity against four bacterial and two fungal pathogens with much higher selectivity index values obtained for A. fumigatus and Cryptococcus neoformans (SI > 30). PFA had a broad spectrum of activity against Enterococcus faecalis and Pseudomonas aeruginosa (SI > 15) and less activity against the two fungal pathogens. In both the cytotoxicity assays and Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, TMA and IGG had no deleterious effect on the different cell types and did not induce mutations in the Ames test. Although the antimicrobial activities of the isolated compounds were not that exciting, the compounds had no cytotoxic activity at the highest concentration (1000 μg/ml) tested against all three cell lines. IGG was the most active against E. coli, S. aureus, A. fumigatus and C. neoformans, exhibiting both antibacterial and antifungal activity with good

  5. Selected lactic acid-producing bacterial isolates with the capacity to reduce Salmonella translocation and virulence gene expression in chickens.

    Directory of Open Access Journals (Sweden)

    Xiaojian Yang

    Full Text Available BACKGROUND: Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control. METHODOLOGY/PRINCIPAL FINDINGS: In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0 and high bile salt (0.3-1.5% and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10(6-7 CFU/chick or phosphate-buffered saline (PBS at 1 day of age followed by Salmonella challenge (10(4 CFU/chick next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1. These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10 in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression. CONCLUSIONS/SIGNIFICANCE: The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in

  6. Relatedness of Listeria monocytogenes Isolates recovered from selected ready-to-eat foods and listeriosis patients in the United States.

    Science.gov (United States)

    Gilbreth, Stefanie Evans; Call, Jeff E; Wallace, F Morgan; Scott, Virginia N; Chen, Yuhuan; Luchansky, John B

    2005-12-01

    Pulsed-field gel electrophoresis and serotyping were performed for 544 isolates of Listeria monocytogenes, including 502 isolates recovered from contaminated samples from 31,705 retail ready-to-eat (RTE) food products and 42 isolates recovered from human cases of listeriosis. The isolates were from Maryland (294 isolates) and California (250 isolates) and were collected in 2000 and 2001. The isolates were placed into 16 AscI pulsogroups (level of relatedness within each group, > or =66%), 139 AscI pulsotypes (levels of relatedness, > or =25% to 100%), and eight serotypes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 4b, 4c, and 4d). The most frequently found pulsotypes belonged to either pulsogroup A (150 food isolates plus 4 clinical isolates) or pulsogroup B (104 food isolates plus 5 clinical isolates). The majority of the 502 food isolates were either serotype 1/2a (298 isolates) or serotype 1/2b (133 isolates), whereas the majority of the 42 clinical isolates were either serotype 1/2a (19 isolates) or serotype 4b (15 isolates). Additionally, 13 clinical isolates displayed pulsotypes also found in food isolates, whereas the remaining 29 clinical isolates displayed 24 unique pulsotypes. These data indicate that most (86%) of the L. monocytogenes subtypes found in the RTE foods sampled belonged to only two serotypes and that 90% of the isolates displayed 73 pulsotypes, with 107 isolates displaying pulsotype 1. These data should help define the distribution and relatedness of isolates found in RTE foods in comparison with isolates that cause listeriosis.

  7. Comparison of ChromID agar and Clostridium difficile selective agar for effective isolation of C. difficile from stool specimens.

    Science.gov (United States)

    Shin, Bo-Moon; Lee, Eun Joo

    2014-01-01

    ChromID Clostridium difficile agar (IDCd; bioMérieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMérieux SA), and multiplex PCR for tcdA, tcdB, and tpi. The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.

  8. Identification of Highly Potent and Selective α-Glucosidase Inhibitors with Antiglycation Potential, Isolated from Rhododendron arboreum

    Directory of Open Access Journals (Sweden)

    Rabia Raza

    2015-01-01

    Full Text Available This study explored antidiabetic potential of eight known pure compounds, isolated from the bark of Rhododendron arboreum. Invitro studies of these compounds against α and β-glucosidases revealed them as very potent and selective inhibitors of α-glucosidase. Compound 7 (3-O-acetylursolic acid was found to be the most potent inhibitor of α-glucosidase with 3.3±0.1µM IC 50 value which was many folds higher than standard inhibitor acarbose. Antiglycation studies of compounds showed that all compounds were also very active antiglycation agents. The studied biological properties of these compounds suggest that they are therapeutically interesting and important tools for treatment of diabetes.

  9. A select bibliography with abstracts of reports related to Waste Isolation Pilot Plant geotechnical studies (1972--1990)

    Energy Technology Data Exchange (ETDEWEB)

    Powers, D.W. [Powers (Dennis W.), Anthony, TX (United States); Martin, M.L. [International Technology, Inc., Las Vegas, NV (United States)

    1993-08-01

    This select bibliography contains 941 entries. Each bibliographic entry contains the citation of a report, conference paper, or journal article containing geotechnical information about the Waste Isolation Pilot Plant (WIPP). The entries cover the period from 1972, when investigation began for a WIPP Site in southeastern New Mexico, through December 1990. Each entry is followed by an abstract. If an abstract or suitable summary existed, it has been included; 316 abstracts were written for other documents. For some entries, an annotation has been provided to clarify the abstract, comment on the setting and significance of the document, or guide the reader to related reports. An index of key words/phrases is included for all entries.

  10. Prevalence and Bacterial Isolates of Mastitis in Dairy Farms in Selected Districts of Eastern Harrarghe Zone, Eastern Ethiopia

    Science.gov (United States)

    Abera, Gerema

    2017-01-01

    The study was conducted from November 2015 to April 2016 to estimate the prevalence of clinical and subclinical mastitis in lactating cows, to assess the associated risk factors, and to isolate the major bacterial pathogens in dairy farms in selected district of Eastern Harrarghe Zone, Eastern Ethiopia. The study was carried out in 384 dairy cows based on data collection, farm visit, animal examination, California mastitis test (CMT), and isolation bacterial pathogens using standard techniques. In the present study the overall mastitis at cow level was 247 (64.3%). The prevalence of clinical and subclinical mastitis and quarter level prevalence for clinical and subclinical mastitis were 12.5% and 51.8% at cow level and 10.7% and 46.4% at quarter level, respectively. Clinically, 101 (6.6%) quarters which belong to 75 (19.5%) animals were found to be with blind teat. In the present study prevalence of mastitis was significantly associated with parity and age (p mastitis particularly the subclinical mastitis was major problem of dairy cows in the area and hence warrants serious attention. PMID:28352648

  11. Poly-γ-Glutamic Acids Contribute to Biofilm Formation and Plant Root Colonization in Selected Environmental Isolates of Bacillus subtilis.

    Science.gov (United States)

    Yu, Yiyang; Yan, Fang; Chen, Yun; Jin, Christopher; Guo, Jian-Hua; Chai, Yunrong

    2016-01-01

    Bacillus subtilis is long known to produce poly-γ-glutamic acids (γ-PGA) as one of the major secreted polymeric substances. In B. subtilis, the regulation of γ-PGA production and its physiological role are still unclear. B. subtilis is also capable of forming structurally complex multicellular communities, or biofilms, in which an extracellular matrix consisting of secreted proteins and polysaccharides holds individual cells together. Biofilms were shown to facilitate B. subtilis-plant interactions. In this study, we show that different environmental isolates of B. subtilis, all capable of forming biofilms, vary significantly in γ-PGA production. This is possibly due to differential regulation of γ-PGA biosynthesis genes. In many of those environmental isolates, γ-PGA seems to contribute to robustness and complex morphology of the colony biofilms, suggesting a role of γ-PGA in biofilm formation. Our evidence further shows that in selected B. subtilis strains, γ-PGA also plays a role in root colonization by the bacteria, pinpointing a possible function of γ-PGA in B. subtilis-plant interactions. Finally, we found that several pathways co-regulate both γ-PGA biosynthesis genes and genes for the biofilm matrix in B. subtilis, but in an opposing fashion. We discussed potential biological significance of that.

  12. An analysis of the binding characteristics of a panel of recently selected ICAM-1 binding Plasmodium falciparum patient isolates.

    Directory of Open Access Journals (Sweden)

    Aymen M Madkhali

    Full Text Available The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1 expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants.

  13. Poly-γ-Glutamic Acids Contribute to Biofilm Formation and Plant Root Colonization in Selected Environmental Isolates of Bacillus subtilis

    Science.gov (United States)

    Yu, Yiyang; Yan, Fang; Chen, Yun; Jin, Christopher; Guo, Jian-Hua; Chai, Yunrong

    2016-01-01

    Bacillus subtilis is long known to produce poly-γ-glutamic acids (γ-PGA) as one of the major secreted polymeric substances. In B. subtilis, the regulation of γ-PGA production and its physiological role are still unclear. B. subtilis is also capable of forming structurally complex multicellular communities, or biofilms, in which an extracellular matrix consisting of secreted proteins and polysaccharides holds individual cells together. Biofilms were shown to facilitate B. subtilis–plant interactions. In this study, we show that different environmental isolates of B. subtilis, all capable of forming biofilms, vary significantly in γ-PGA production. This is possibly due to differential regulation of γ-PGA biosynthesis genes. In many of those environmental isolates, γ-PGA seems to contribute to robustness and complex morphology of the colony biofilms, suggesting a role of γ-PGA in biofilm formation. Our evidence further shows that in selected B. subtilis strains, γ-PGA also plays a role in root colonization by the bacteria, pinpointing a possible function of γ-PGA in B. subtilis–plant interactions. Finally, we found that several pathways co-regulate both γ-PGA biosynthesis genes and genes for the biofilm matrix in B. subtilis, but in an opposing fashion. We discussed potential biological significance of that. PMID:27891125

  14. Isolating GaSb membranes grown metamorphically on GaAs substrates using highly selective substrate removal etch processes

    Energy Technology Data Exchange (ETDEWEB)

    Lavrova, Olga [Univ. of New Mexico, Albuquerque, NM (United States). Dept. of Electrical and Computer Engineering. Center for High Technology Materials; Balakrishnan, Ganesh [Univ. of New Mexico, Albuquerque, NM (United States). Dept. of Electrical and Computer Engineering. Center for High Technology Materials

    2017-02-24

    The etch rates of NH4OH:H2O2 and C6H8O7:H2O2 for GaAs and GaSb have been investigated to develop a selective etch for GaAs substrates and to isolate GaSb epilayers grown on GaAs. The NH4OH:H2O2 solution has a greater etch rate differential for the GaSb/GaAs material system than C6H8O7:H2O2 solution. The selectivity of NH4OH:H2O2 for GaAs/GaSb under optimized etch conditions has been observed to be as high as 11471 ± 1691 whereas that of C6H8O7:H2O2 has been measured up to 143 ± 2. The etch contrast has been verified by isolating 2 μm thick GaSb epi-layers that were grown on GaAs substrates. GaSb membranes were tested and characterized with high-resolution X-Ray diffraction (HR-XRD) and atomic force microscopy (AFM).

  15. Selection and Characterization of a Lysine Yielding Mutant of Corynebacterium glutamicum - a Soil Isolate from Pakistan

    Directory of Open Access Journals (Sweden)

    Habib-ur-Rehman§٭, Abdul Hameed and Safia Ahmed

    2012-01-01

    Full Text Available L-lysine is the second limiting amino acid for poultry and supplemented in broiler feed for optimal performance. Lysine can be produced by inducing mutation in glutamate producing bacteria. The study was conducted to enhance lysine production from a local strain of Corynebacterium glutamicum. The bacterium was mutated by exposure to UV. Mutants resistant to s-2-aminoethyle L-cystein (AEC and showing auxotrophy for L-homoserine were screened for lysine production qualitatively and quantitatively. A mutant showing highest production of lysine (8.2 mg/mL was selected for optimization of physical and nutritional parameters for maximum production of lysine in shake flask. An initial pH 7.6, 30˚C temperature, 300 rpm and 60 h incubation time were the optimized values of physical requirements. Cane molasses and corn starch hydrolysate were required at 15% (w/v in the fermentation media which provided around 9% total sugars to produce maximum lysine (17 to 18 mg/mL. When amonium sulphate was used at 3.5% (w/v level in molasses or corn starch hydrolysate based fermentation media, production of lysine slightly increased above 18 mg/mL. It is concluded that industrial by products like cane molasses, corn steep liquor, and corn starch hydrolysate can be used as carbon and organic nitrogen sources in fermentation medium for scale up process of lysine production and this lysine enriched broth may be used in broiler feed later. However, more potent lysine producing mutant and additional in vivo trials would be required to commercialize this product.

  16. Industrial application of selected lactic acid bacteria isolated from local semolinas for typical sourdough bread production.

    Science.gov (United States)

    Corona, Onofrio; Alfonzo, Antonio; Ventimiglia, Giusi; Nasca, Anna; Francesca, Nicola; Martorana, Alessandra; Moschetti, Giancarlo; Settanni, Luca

    2016-10-01

    strict relation was found among the productions carried out by W. cibaria strains. This study proved the suitability of the selected strains of L. citreum and W. cibaria for industrial-scale level applications in sourdough bread production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. A Multidimensional System for Phosphopeptide Analysis Using TiO{sub 2} Enrichment and Ion-exchange Chromatography with Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Kun; Yoo, Jisun; Kim, Eunmin; Kim, Jin Young; Kim, Young Hwan; Yoo, Jong Shin [Korea Basic Science Institute, Ochang (Korea, Republic of); Oh, Han Bin [Sogang Univ., Seoul (Korea, Republic of)

    2012-10-15

    Although offline enrichment of phosphorylated peptides is widely used, enrichment for phosphopeptides using TiO{sub 2} is often performed manually, which is labor-intensive and can lead to irreproducible results. To address the problems associated with offline enrichment and to improve the effectiveness of phosphopeptide detection, we developed an automated online enrichment system for phosphopeptide analysis. A standard protein mixture comprising BSA, fetuin, crystalline, α-casein and β-casein, and ovalbumin was assessed using our new system. Our multidimensional system has four main parts: a sample pump, a 20-mm TiO{sub 2}-based column, a weak anion-exchange, and a strong cation-exchange (2:1 WAX:SCX) separation column with LC/MS. Phosphorylated peptides were successfully detected using the TiO{sub 2}-based online system with little interference from nonphosphorylated peptides. Our results confirmed that our online enrichment system is a simple and efficient method for detecting phosphorylated peptides.

  18. Incarceration of Higher-Order Fullerenes within Cyclotriveratrylene-Based Hemicarcerands Allows Selective Isolation of C76 , C78 , and C84 from a Commercial Fullerene Mixture.

    Science.gov (United States)

    Liu, Kuang-Shun; Li, Ming-Jhe; Lai, Chien-Chen; Chiu, Sheng-Hsien

    2016-11-21

    Size-complementary cyclotriveratrylene (CTV)-based hosts can incarcerate C76 , C78 , and C84 , thus allowing the selective isolation of these higher-order fullerenes from a commercially available mixture of fullerenes. The hemicarceplexes, formed after the encapsulation of the size-complementary fullerenes within the hosts, are isolated by column chromatography and released at elevated temperature, thereby leading to the isolation of C76 /C78 and C84 in good purities (up to 95 and 88 %, respectively). © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Isolated Calyx Mistaken for a Cyst: Inappropriately Performed Catheter-Directed Sclerotherapy and Safe Removal of the Catheter After Selective Embolization

    Energy Technology Data Exchange (ETDEWEB)

    Gwak, Jng Won, E-mail: jjungwonie@hanmail.net; Lee, Seung Hwa, E-mail: gareureung@daum.net; Chung, Hwan Hoon, E-mail: chungmic@korea.ac.kr; Je, Bo Kyung, E-mail: purity21@hanmail.net; Yeom, Suk kyu, E-mail: pagoda20@hanmail.net [Korea University College of Medicine, Department of Radiology, Ansan Hospital (Korea, Republic of); Sung, Deuk Jae, E-mail: urora@korea.ac.kr [Korea University College of Medicine, Department of Radiology, Anam Hospital (Korea, Republic of)

    2015-02-15

    We present a case of isolated calyx that was mistaken for a large cyst. A 47-year-old woman was referred for sclerotherapy of a large cystic lesion on her left kidney. Computed tomography (CT) and ultrasound showed that the cystic lesion was a large cyst. We noticed that the cystic lesion was not a typical simple cyst, even after two sessions of catheter-mediated sclerotherapy. Isolated calyx was presumed by medical history review and was confirmed by aspirated fluid analysis and far delayed-phase CT after intravenous contrast injection. We performed meticulous selective arterial embolization for an isolated calyx and inserted a catheter that could be removed without complication.

  20. Hybridization, natural selection, and evolution of reproductive isolation: a 25-years survey of an artificial sympatric area between two mosquito sibling species of the Aedes mariae complex.

    Science.gov (United States)

    Urbanelli, Sandra; Porretta, Daniele; Mastrantonio, Valentina; Bellini, Romeo; Pieraccini, Giuseppe; Romoli, Riccardo; Crasta, Graziano; Nascetti, Giuseppe

    2014-10-01

    Natural selection can act against maladaptive hybridization between co-occurring divergent populations leading to evolution of reproductive isolation among them. A critical unanswered question about this process that provides a basis for the theory of speciation by reinforcement, is whether natural selection can cause hybridization rates to evolve to zero. Here, we investigated this issue in two sibling mosquitoes species, Aedes mariae and Aedes zammitii, that show postmating reproductive isolation (F1 males sterile) and partial premating isolation (different height of mating swarms) that could be reinforced by natural selection against hybridization. In 1986, we created an artificial sympatric area between the two species and sampled about 20,000 individuals over the following 25 years. Between 1986 and 2011, the composition of mating swarms and the hybridization rate between the two species were investigated across time in the sympatric area. Our results showed that A. mariae and A. zammitii have not completed reproductive isolation since their first contact in the artificial sympatric area. We have discussed the relative role of factors such as time of contact, gene flow, strength of natural selection, and biological mechanisms causing prezygotic isolation to explain the observed results. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

  1. Ionic liquid templated porous nano-TiO2 particles for the selective isolation of cytochrome c.

    Science.gov (United States)

    Meng, Hao; Chen, Xu-Wei; Wang, Jian-Hua

    2010-09-24

    Porous nano-titanium dioxide (TiO(2)) particles with anatase framework were prepared by using an effective template of hydrophobic ionic liquid, i.e., N, N-bis [2-methylbutyl] imidazolium hexafluorophosphate ([PPim][PF(6)]). The nano-TiO(2) particles were characterized with TEM and BET, resulting in the distribution of the pore diameters centering at 7.3 nm and 26.2 nm, attributed to the template effect of the ionic liquid and the aggregation of the TiO(2) particles, respectively. A much improved surface area of 68.31 m(2) g(-1) and a pore volume of 0.2814 cm(3) g(-1) were obtained with respect to 15.16 m(2) g(-1) and 0.0402 cm(3) g(-1) achieved by the non-templated nano-TiO(2) counterpart. The porous nano-TiO(2) particles were used as adsorbent for selective adsorption and isolation of cytochrome c (cyt-c) at certain conditions. An adsorption efficiency of 87% was achieved for 150 microg ml(-1) cyt-c in 1.0 ml of sample solution at pH 9.0 by using 2.0 mg of the nano-TiO(2) particles, in comparison to 30% obtained by the non-templated nano-TiO(2). In addition, an adsorption capacity of 112.6 microg mg(-1) was derived for the porous nano-TiO(2) with respect to 25.1 microg mg(-1) for the normal ones. The absorbed cyt-c could be readily recovered by stripping with a 0.1 mol l(-1) Na(3)PO(4) solution, giving rise to a recovery of ca. 90%. The porous nano-TiO(2) particles have been used for the isolation of cyt-c from human whole blood, achieving satisfactory results by assay with SDS-PAGE.

  2. Screening and selection of most potent diazotrophic cyanobacterial isolate exhibiting natural tolerance to rice field herbicides for exploitation as biofertilizer.

    Science.gov (United States)

    Singh, Surendra; Datta, Pallavi

    2006-01-01

    Periodic applications of heavy dosages of herbicides in modern rice-agriculture are a necessary evil for obtaining high crop productivity. Such herbicides are not only detrimental to weeds but biofertilizer strains of diazotrophic cyanobacteria also. It is therefore, essential to screen and select such biofertilizer strains of diazotrophic cyanobacteria exhibiting natural tolerance to common rice-field herbicides that can be further improved by mutational techniques to make biofertilizer technology a viable one. Therefore, efforts have been made to screen five dominant diazotrophic cyanobacterial forms e.g. filamentous heterocystous Nostoc punctiforme , Nostoc calcicola , Anabaena variabilis and unicellular Gloeocapsa sp. and Aphanocapsa sp. along with standard laboratory strain Nostoc muscorum ISU against increasing concentrations (0-100 mg l(-1) of four commercial grade common rice-field herbicides i.e. Arozin, Butachlor, Alachlor and 2,4-D under diazotrophic growth conditions. The lethal and IGC(50) concentrations for all four herbicides tested were found highest for A. variabilis as compared to other test cyanobacteria. The lowest reduction in chlorophyll a content, photosynthetic oxygen evolution, and N(2)-fixation was found in A. variabilis as compared to other rice field isolates and standard laboratory strain N. muscorum ISU. On the basis of prolong survival potential and lowest reductions in vital metabolic activities tested at IGC(50) concentration of four herbicides, it is concluded that A. variabilis is the most potent and promising cyanobacterial isolate as compared with other forms. This could be further improved by mutational techniques for exploitation as most potential and viable biofertilizer strain.

  3. Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars.

    Science.gov (United States)

    Isnard, M; Granier, M; Frutos, R; Reynaud, B; Peterschmitt, M

    1998-12-01

    Three maize streak virus (MSV) isolates were derived from an MSV population used to assess the response to infection of maize cultivars. Isolate SP1 was obtained from this population through short acquisition and inoculation periods (1 and 5 min, respectively), using a single Cicadulina mbila vector. Isolate SP2 was derived from SP1 after transmission to a wild perennial host (Coix lacryma-jobi), on which it was maintained for about 4 years without insect transmission. Isolate N2A, the most pathogenic isolate, was obtained from the initial population after serial passages on almost completely resistant inbred maize lines. The complexity of each isolate was analysed by RFLP analysis and sequencing based on 120 SP1 clones, 36 SP2 clones and 40 N2A clones. All three isolates were composed of different but related clones, consistent with a quasispecies structure. The mutations were distributed throughout the genome. Mutation frequencies, based on all available sequences, were 3.8 x 10(-4) for SP1, 10.5 x 10(-4) for SP2 and 6.9 x 10(-4) for N2A. As expected from the bottleneck selection step, the intra-isolate variability of SP1 was relatively low. Comparison between SP1 and SP2 showed that SP1 heterogeneity increased during maintenance on the wild host. Furthermore, the consensus sequences of SP1 and SP2 differed by two non-synonymous substitutions in the complementary sense gene repA. N2A had a relatively low degree of heterogeneity, but was composed of several sub-populations. The results reflect the influence of the mode of selection of MSV isolates on their quasispecies organization, i.e. distribution of variants, and master sequence.

  4. Selection against recombinant hybrids maintains reproductive isolation in hybridizing Populus species despite F1 fertility and recurrent gene flow.

    Science.gov (United States)

    Christe, Camille; Stölting, Kai N; Bresadola, Luisa; Fussi, Barbara; Heinze, Berthold; Wegmann, Daniel; Lexer, Christian

    2016-06-01

    Natural hybrid zones have proven to be precious tools for understanding the origin and maintenance of reproductive isolation (RI) and therefore species. Most available genomic studies of hybrid zones using whole- or partial-genome resequencing approaches have focused on comparisons of the parental source populations involved in genome admixture, rather than exploring fine-scale patterns of chromosomal ancestry across the full admixture gradient present between hybridizing species. We have studied three well-known European 'replicate' hybrid zones of Populus alba and P. tremula, two widespread, ecologically divergent forest trees, using up to 432 505 single-nucleotide polymorphisms (SNPs) from restriction site-associated DNA (RAD) sequencing. Estimates of fine-scale chromosomal ancestry, genomic divergence and differentiation across all 19 poplar chromosomes revealed strikingly contrasting results, including an unexpected preponderance of F1 hybrids in the centre of genomic clines on the one hand, and genomically localized, spatially variable shared variants consistent with ancient introgression between the parental species on the other. Genetic ancestry had a significant effect on survivorship of hybrid seedlings in a common garden trial, pointing to selection against early-generation recombinants. Our results indicate a role for selection against recombinant genotypes in maintaining RI in the face of apparent F1 fertility, consistent with the intragenomic 'coadaptation' model of barriers to introgression upon secondary contact. Whole-genome resequencing of hybridizing populations will clarify the roles of specific genetic pathways in RI between these model forest trees and may reveal which loci are affected most strongly by its cyclic breakdown. © 2016 John Wiley & Sons Ltd.

  5. Comparative evaluation of the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol-containing chewing gum on salivary flow rate, pH and buffering capacity in children: Anin vivostudy.

    Science.gov (United States)

    Hegde, Rahul J; Thakkar, Janhavi B

    2017-01-01

    This study aimed to compare and evaluate the changes in the salivary flow rate, pH, and buffering capacity before and after chewing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and xylitol-containing chewing gums in children. Sixty children aged between 8 and 12 years were selected for the study. They were randomly divided into Group 1 (CPP-ACP chewing gum) and Group 2 (xylitol-containing chewing gum) comprising thirty children each. Unstimulated and stimulated saliva samples at 15 and 30 min interval were collected from all children. All the saliva samples were estimated for salivary flow rate, pH, and buffering capacity. Significant increase in salivary flow rate, pH, and buffering capacity from baseline to immediately after spitting the chewing gum was found in both the study groups. No significant difference was found between the two study groups with respect to salivary flow rate and pH. Intergroup comparison indicated a significant increase in salivary buffer capacity in Group 1 when compared to Group 2. Chewing gums containing CPP-ACP and xylitol can significantly increase the physiochemical properties of saliva. These physiochemical properties of saliva have a definite relation with caries activity in children.

  6. Comparative evaluation of the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP and xylitol-containing chewing gum on salivary flow rate, pH and buffering capacity in children: An in vivo study

    Directory of Open Access Journals (Sweden)

    Rahul J Hegde

    2017-01-01

    Full Text Available Aim: This study aimed to compare and evaluate the changes in the salivary flow rate, pH, and buffering capacity before and after chewing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP and xylitol-containing chewing gums in children. Materials and Methods: Sixty children aged between 8 and 12 years were selected for the study. They were randomly divided into Group 1 (CPP-ACP chewing gum and Group 2 (xylitol-containing chewing gum comprising thirty children each. Unstimulated and stimulated saliva samples at 15 and 30 min interval were collected from all children. All the saliva samples were estimated for salivary flow rate, pH, and buffering capacity. Results: Significant increase in salivary flow rate, pH, and buffering capacity from baseline to immediately after spitting the chewing gum was found in both the study groups. No significant difference was found between the two study groups with respect to salivary flow rate and pH. Intergroup comparison indicated a significant increase in salivary buffer capacity in Group 1 when compared to Group 2. Conclusion: Chewing gums containing CPP-ACP and xylitol can significantly increase the physiochemical properties of saliva. These physiochemical properties of saliva have a definite relation with caries activity in children.

  7. Antibacterial Effects of Origanum vulgare Essence Against Multidrug-Resistant Acinetobacter baumannii Isolated From Selected Hospitals of Tehran, Iran

    Directory of Open Access Journals (Sweden)

    Saghi

    2015-02-01

    Full Text Available Background Infection due to Acinetobacter baumannii has become a significant challenge to modern healthcare systems. The rapid emergence and global dissemination of A. baumannii as a major nosocomial pathogen is remarkable and it demonstrates its successful adaptation to the 21st century hospital environment. Recent studies have discussed about essential oil of Origanum vulgare against a range of bacteria, including various species of Staphylococcus, Pseudomonas, Bacillus and Escherichia coli. Objectives The present study aimed to investigate the inhibitory effects O. vulgare essence against multidrug-resistant (MDR strains of A. baumannii from selected hospitals in Tehran, Iran. Materials and Methods This oil was obtained using the hydrodistillation method and analyzed by gas chromatography mass spectrography (GC/MS. The antimicrobial activity against MDR isolates was achieved using disc diffusion method and macro-broth dilution assay. Results Analysis of the essential oil revealed the presence of pulegone (68.59% piperitone (7.8%, piperitenone (7.8%, 1, 8-cineole (1.3%, and carvacrol (1.6% as the major components. The results showed a significant activity against MDR A. baumannii with inhibition zones and minimal inhibitory concentration values in the ranges of 7-15 mm and 20-35 µL/mL respectively. Conclusions This investigation showed that the essence oil of O. vulgare had a potent antimicrobial activity against MDR A. baumannii. Further research is required to evaluate the practical values of therapeutic applications.

  8. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    Purpose. A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture...... for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1...... medium (SuperPolymyxin). Methodology. The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth...

  9. Different Patterns of pfcrt and pfmdr1 Polymorphisms in P. falciparum Isolates from Nigeria and Brazil: The Potential Role of Antimalarial Drug Selection Pressure

    Science.gov (United States)

    Gbotosho, Grace O.; Folarin, Onikepe A.; Bustamante, Carolina; Pereira da Silva, Luis Hildebrando; Mesquita, Elieth; Sowunmi, Akintunde; Zalis, Mariano G.; Oduola, Ayoade M. J.; Happi, Christian T.

    2012-01-01

    The effect of antimalarial drug selection on pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from two distinct geographical locations was determined in 70 and 18 P. falciparum isolates from Nigeria and Brazil, respectively, using nested polymerase chain reaction and direct DNA sequencing approaches. All isolates from Brazil and 72% from Nigeria harbored the mutant SVMNT and CVIET pfcrt haplotype, respectively. The pfcrt CVMNT haplotype was also observed in (7%) of the Nigerian samples. One hundred percent (100%) and 54% of the parasites from Brazil and Nigeria, respectively, harbored wild-type pfmdr1Asn86. We provide first evidence of emergence of the CVMNT haplotype in West Africa. The high prevalence of pfcrt CVIET and SVMNT haplotypes in Nigeria and Brazil, respectively, is indicative of different selective pressure by chloroquine and amodiaquine. Continuous monitoring of pfcrt SVMNT haplotype is required in endemic areas of Africa, where artesunate-amodiaquine combination is used for treatment of acute uncomplicated malaria. PMID:22302850

  10. Selection of colistin-resistant Acinetobacter baumannii isolates in postneurosurgical meningitis in an intensive care unit with high presence of heteroresistance to colistin.

    Science.gov (United States)

    Rodriguez, Carlos Hernan; Hernan, Rodriguez Carlos; Bombicino, Karina; Karina, Bombicino; Granados, Gabriela; Gabriela, Granados; Nastro, Marcela; Marcela, Nastro; Vay, Carlos; Carlos, Vay; Famiglietti, Angela; Angela, Famiglietti

    2009-10-01

    Colistin heteroresistance in Acinetobacter baumannii (Ab) has been reported, but the clinical impact and the antimicrobial treatment have not been established yet. We observed the selection intratreatment with colistin of Ab colistin-resistant strains from a colistin-heteroresistant isolate in one patient with postneurosurgical meningitis. The presence and the genetic relationship of heteroresistant Ab isolates from intensive care units (ICUs) obtained in the same period of the case report were analyzed. Twenty-eight isolates from patients admitted to the ICUs of an Argentinian university hospital during June to December 2004 were evaluated. Genomoespecie was determined by amplified ribosomal DNA restriction analysis, and genetic similarity among the strains was determined by pulsed-field electrophoresis. Colistin heteroresistance was observed in 46, 4% of these isolates. The majority belonged to clones previously identified as I and III.

  11. Combining casein phosphopeptide-amorphous calcium phosphate with fluoride: synergistic remineralization potential of artificially demineralized enamel or not?

    Science.gov (United States)

    Elsayad, Iman; Sakr, Amal; Badr, Yahia

    2009-07-01

    Recaldent is a product of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). The remineralizing potential of CPP-ACP per se, or when combined with 0.22% Fl gel on artificially demineralized enamel using laser florescence, is investigated. Mesial surfaces of 15 sound human molars are tested using a He-Cd laser beam at 441.5 nm with 18-mW power as an excitation source on a suitable setup based on a Spex 750-M monochromator provided with a photomultiplier tube (PMT) for detection of collected autofluorescence from sound enamel. Mesial surfaces are subjected to demineralization for ten days. The spectra from demineralized enamel are measured. Teeth are divided into three groups according to the remineralizing regimen: group 1 Recaldent per se, group 2 Recaldent combined with fluoride gel and ACP, and group 3 artificial saliva as a positive control. After following these protocols for three weeks, the spectra from the remineralized enamel are measured. The spectra of enamel autofluorescence are recorded and normalized to peak intensity at about 540 nm to compare spectra from sound, demineralized, and remineralized enamel surfaces. A slight red shift occurred in spectra from demineralized enamel, while a blue shift may occur in remineralized enamel. Group 2 shows the highest remineralizing potential. Combining fluoride and ACP with CPP-ACP can give a synergistic effect on enamel remineralization.

  12. Clinical evaluation of remineralization potential of casein phosphopeptide amorphous calcium phosphate nanocomplexes for enamel decalcification in orthodontics.

    Science.gov (United States)

    Wang, Jun-xiang; Yan, Yan; Wang, Xiu-jing

    2012-11-01

    Enamel decalcification in orthodontics is a concern for dentists and methods to remineralize these lesions are the focus of intense research. The aim of this study was to evaluate the remineralizing effect of casein phosphopeptide amorphous calcium phosphate (CPP-ACP) nanocomplexes on enamel decalcification in orthodontics. Twenty orthodontic patients with decalcified enamel lesions during fixed orthodontic therapy were recruited to this study as test group and twenty orthodontic patients with the similar condition as control group. GC Tooth Mousse, the main component of which is CPP-ACP, was used by each patient of test group every night after tooth-brushing for six months. For control group, each patient was asked to brush teeth with toothpaste containing 1100 parts per million (ppm) of fluoride twice a day. Standardized intraoral images were taken for all patients and the extent of enamel decalcification was evaluated before and after treatment over this study period. Measurements were statistically compared by t test. After using CPP-ACP for six months, the enamel decalcification index (EDI) of all patients had decreased; the mean EDI before using CPP-ACP was 0.191 ± 0.025 and that after using CPP-ACP was 0.183 ± 0.023, the difference was significant (t = 5.169, P 0.05). CPP-ACP can effectively improve the demineralized enamel lesions during orthodontic treatment, so it has some remineralization potential for enamel decalcification in orthodontics.

  13. Selection, assessment of virulence to Alphitobius diaperinus, and Pr1 enzyme production of Beauveria bassiana (Bals. Vuill. isolates cultured at stress temperatures

    Directory of Open Access Journals (Sweden)

    Kelly Christiane Constanski

    2015-12-01

    Full Text Available The entomopathogenic fungus Beauveria bassiana is a promising agent for use in insect control. Its pathogenic activity, as well as other factors such as temperature that can interfere with its development, should be assessed, thus, establishing the foundations for B. bassiana use in biological control programs. The objective of this study was to select and induce tolerance of B. bassiana isolates to high and low temperatures and to assess their virulence before and after exposure to those temperatures. A pre-selection test was performed, in which the tolerance of isolates to stress temperatures was tested and compared to the ideal growth temperature of 25 °C for this organism. For the isolates/temperature combinations resulting in growth, conidia germination and colony-forming units (CFUs were assessed. The isolates Unioeste 4 and Unioeste 40 exhibited >95% germinated conidia at 16 and 31 °C. Thereafter, they underwent four consecutive passages at maximum and minimum tolerated temperatures (10 and 37 °C. A significant difference in germination was observed between the two isolates at all temperatures tested. More CFUs were observed for Unioeste 4 compared to Unioeste 40 at all temperatures, and in the case of the latter, there was no difference in CFU formation at 10 and 25 °C. For both isolates, decreased vegetative growth was observed at 37 °C. Recovery of virulence was observed in both isolates, as determined by insect mortality. No relationship was observed between production of the enzyme Pr1 and the virulence of the isolates.

  14. Coupling of Phosphate-Imprinted Mesoporous Silica Nanoparticles-Based Selective Enrichment with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry for Highly Efficient Analysis of Protein Phosphorylation.

    Science.gov (United States)

    Chen, Yang; Li, Daojin; Bie, Zijun; He, Xinpei; Liu, Zhen

    2016-01-19

    Protein phosphorylation is a major post-translational modification and represents a ubiquitous mechanism for the cellular signaling of many different biological processes. Selective enrichment of phosphopeptides from the complex biological samples is a key step for the mass spectrometric (MS) analysis of protein phosphorylation. Herein, we present phosphate-imprinted mesoporous silica nanoparticles (MSNs) as an ideal sorbent for selective enrichment of phosphopeptides and an off-line combination with matrix-asisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for highly efficient analysis of protein phosphorylation. The phosphate-imprinted MSNs were prepared according to a newly reported strategy called dual-template docking oriented molecular imprinting (DTD-OMI). The prepared molecularly imprinted mesoporous material exhibited several significant merits, such as excellent selectivity toward phosphopeptides, tolerance to interference, fast binding equilibrium, and large binding capacity, which made the molecularly imprinted mesoporous material an ideal sorbent for selective enrichment of phosphopeptides. Using β-casein as a representative phosphoprotein, highly efficient phosphorylation analysis by the off-line platform was verified. Phosphorylation analysis of a nonfat milk sample was also well demonstrated. Because of their highly desirable properties, the phosphate-imprinted MSNs could find more applications in the analysis of protein phosphorylation.

  15. Isolation of Individual Egg Cells and Zygotes in Alstroemeria Followed by Manual Selection with a Microcapillary-connected Micropump

    OpenAIRE

    HOSHINO, YOICHIRO; MURATA, NAHO; SHINODA, KOICHI

    2006-01-01

    Aims: To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. Scope: We attempted to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these o...

  16. Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump.

    Science.gov (United States)

    Hoshino, Yoichiro; Murata, Naho; Shinoda, Koichi

    2006-06-01

    To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary.

  17. Determining the prevalence of SCCmec polymorphism, virulence and antibiotic resistance genes among methicillin-resistant Staphylococcus aureus (MRSA) isolates collected from selected hospitals in west of Iran.

    Science.gov (United States)

    Taherikalani, Morovat; Mohammadzad, Mohammad Reza; Soroush, Setareh; Maleki, Mohammad Hossein; Azizi-Jalilian, Farid; Pakzad, Iraj; Sadeghifard, Nourkhoda; Asadollahi, Parisa; Emaneini, Mohammad; Monjezi, Aazam; Alikhani, Mohammad Yousef

    2016-04-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens worldwide and compared to other staphylococcal species that are associated with higher mortality rate. A total of 500 Staphylococcus spp. was collected from selected hospitals in Ilam, Kermanshah, Khorram Abad and Hamadan cities and, via phenotypic and genotypic methods, was assessed to find MRSA. The presence or absence of prevalent antibiotic resistance genes and virulence genes was evaluated among MRSA isolates, using polymerase chain reaction (PCR) method, and then the SCCmec typing of these isolates was assayed by multiplex PCR. A total of 372 (74.4%) Stapylococcus spp. isolates were identified as S. aureus, among which 200 (53.8%) possessed the mecA gene and were distinguished as MRSA. All of MRSA isolates contained blaZ gene. The frequency of ermA and ermC genes among erythromycin-resistant MRSA isolates was 21.6% and 66.7%, respectively. The frequency of the virulence genes eta, hla and sea among MRSA isolates was 10%, 80.5% and 100%, respectively. SCCmec type IV accounted for 30.6% of the MRSA isolates and SCCmec type III, SCCmec type II and SCCmec type I accounted for 30%, 22% and 17.5% of the isolates, respectively. The antibiotic resistance genes and the virulence genes of blaZ, hla, sea, eta and ermC had high frequencies among the MRSA isolates. This study showed that the antibiotic resistance genes had higher frequencies among SCCmec types I and IV, which confirms the previous reports in this field.

  18. Coupling of metal-organic frameworks-containing monolithic capillary-based selective enrichment with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for efficient analysis of protein phosphorylation.

    Science.gov (United States)

    Li, Daojin; Yin, Danyang; Chen, Yang; Liu, Zhen

    2017-05-19

    Protein phosphorylation is a major post-translational modification, which plays a vital role in cellular signaling of numerous biological processes. Mass spectrometry (MS) has been an essential tool for the analysis of protein phosphorylation, for which it is a key step to selectively enrich phosphopeptides from complex biological samples. In this study, metal-organic frameworks (MOFs)-based monolithic capillary has been successfully prepared as an effective sorbent for the selective enrichment of phosphopeptides and has been off-line coupled with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for efficient analysis of phosphopeptides. Using š-casein as a representative phosphoprotein, efficient phosphorylation analysis by this off-line platform was verified. Phosphorylation analysis of a nonfat milk sample was also demonstrated. Through introducing large surface areas and highly ordered pores of MOFs into monolithic column, the MOFs-based monolithic capillary exhibited several significant advantages, such as excellent selectivity toward phosphopeptides, superb tolerance to interference and simple operation procedure. Because of these highly desirable properties, the MOFs-based monolithic capillary could be a useful tool for protein phosphorylation analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Quantification of Competing H3PO4 Versus HPO3 + H2O Neutral Losses from Regioselective 18O-Labeled Phosphopeptides

    Science.gov (United States)

    Cui, Li; Yapici, Ipek; Borhan, Babak; Reid, Gavin E.

    2014-01-01

    Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H3PO4 from the phosphorylated residue and the combined losses of HPO3 and H2O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS3 for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective 18O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the -100 Da (-H3PO3 18O) versus -98 Da (-[HPO3 + H2O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.

  20. A study using demographic data of genetic drift and natural selection in an isolated Mediterranean community: Bayárcal (La Alpujarra, south-east Spain).

    Science.gov (United States)

    Luna, F; Tarelho, A R; Camargo, A M; Alonso, V

    2011-07-01

    Natural selection and genetic drift are two evolutionary mechanisms that can be analysed in human populations using their fertility and mortality patterns, and their reproductive size and isolation, respectively. This paper analyses the models of natural selection and genetic drift in Bayárcal, south-east Spain, and compares them with the observed models in the rest of the Alpujarran region. Demographic data were obtained from a sample of 77 families (48.45% of the population, with 547 inhabitants). The genetic drift and natural selection action was evaluated with the Coefficient of Breeding Isolation (CBI of Lasker and Kaplan) and Crow's index, respectively. The CBI (23.23/12.61) suggests that genetic drift is near to acting, and Crow's index (I=0.58) is slightly higher than that observed in the rest of La Alpujarra. Although the reproductive isolation of Bayárcal is not effective enough for genetic drift to act, it is near when marital migrants inside the Bayárcal valley are considered as a native population. The natural selection pattern is not different from that of the rest of La Alpujarra, but it tends towards the model of developing communities, where the demographic transition has not yet begun. © Cambridge University Press, 2011

  1. Casein phosphopeptide-amorphous calcium phosphate incorporated into sugar confections inhibits the progression of enamel subsurface lesions in situ.

    Science.gov (United States)

    Walker, G D; Cai, F; Shen, P; Adams, G G; Reynolds, C; Reynolds, E C

    2010-01-01

    Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been demonstrated to exhibit anticariogenic activity in randomized, controlled clinical trials of sugar-free gum and a tooth cream. Two randomized, double-blind, crossover studies were conducted to investigate the potential of CPP-ACP added to hard candy confections to slow the progression of enamel subsurface lesions in an in situ model. The confections studied were: (1) control sugar (65% sucrose + 33% glucose syrup); (2) control sugar-free; (3) sugar + 0.5% (w/w) CPP-ACP; (4) sugar + 1.0% (w/w) CPP-ACP; (5) sugar-free + 0.5% (w/w) CPP-ACP. Participants (10 and 14 in study 1 and 2) wore a removable palatal appliance containing enamel half-slabs with subsurface lesions, except for meals and oral hygiene procedures, and consumed 1 confection 6 times a day for 10 days. The enamel half-slabs were inset to allow the development of plaque on the enamel surface. Participants rested for 1 week before crossing over to another confection. The appliances were stored in a humid container at 37 degrees C when not in the mouth. After each treatment period, the enamel half-slabs were removed, paired with their demineralized control half-slabs, embedded, sectioned and then analysed using transverse microradiography. In both studies consumption of the control sugar confection resulted in significant demineralization (progression) of the enamel subsurface lesions. However, consumption of the sugar confections containing CPP-ACP did not result in lesion progression, but in fact in significant remineralization (regression) of the lesions. Remineralization by consumption of the sugar + 1.0% CPP-ACP confection was significantly greater than that obtained with the sugar-free confection. Copyright 2010 S. Karger AG, Basel.

  2. Casein Phosphopeptide-Amorphous Calcium Phosphate Reduces Streptococcus mutans Biofilm Development on Glass Ionomer Cement and Disrupts Established Biofilms.

    Science.gov (United States)

    Dashper, Stuart G; Catmull, Deanne V; Liu, Sze-Wei; Myroforidis, Helen; Zalizniak, Ilya; Palamara, Joseph E A; Huq, N Laila; Reynolds, Eric C

    2016-01-01

    Glass ionomer cements (GIC) are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm) formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge.

  3. Casein Phosphopeptide-Amorphous Calcium Phosphate Reduces Streptococcus mutans Biofilm Development on Glass Ionomer Cement and Disrupts Established Biofilms.

    Directory of Open Access Journals (Sweden)

    Stuart G Dashper

    Full Text Available Glass ionomer cements (GIC are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge.

  4. Effect of casein phosphopeptide-amorphous calcium phosphate on the flexural strength of enamel-dentin complex following extracoronal bleaching

    Directory of Open Access Journals (Sweden)

    Diatri Nari Ratih

    2015-03-01

    Full Text Available Background: Bleaching can affect the mechanical properties of enamel-dentin complex, such as flexural strength. Casein phosphopeptide-amorphus calcium phosphate (CPP-ACP is often used following bleaching treatment to reduce hypersensitivity and to increase demineralization of tooth. Purpose: The purpose of the study was to investigate the effect of CPP-ACP on the flexural strength of enamel-dentin complex following extracoronal bleaching. Methods: Forty-eight enamel-dentin plates (size 8 x 2 x 3 mm were randomly assigned into 6 groups, each consisted of 8 samples. Group 1, no bleaching and immersed in artificial saliva. Group 2, no bleaching, CPP-ACP application only. Group 3, bleaching using 15% carbamide peroxide. Group 4, similar to group 3, except application of CPP-ACP for the times between bleaching. Group 5, bleaching with 40% hydrogen peroxide. Group 6, similar to group 5, except application of CPP-ACP for the times between bleaching. Flexural strength of each enamel-dentin plate was tested by threepoint bending test using universal testing machine. Results: The results showed that 15% carbamide peroxide and 40% hydrogen peroxide significantly reduced flexural strength of enamel-dentin (216.25±26.44 MPa and 206.67±32.07 MPa respectively. Conversely, application of CPP-ACP following both bleachings increased flexural strength (266.75± 28.27MPa and 254.58±36.59 MPa respectively. A two-way Anova revealed that extracoronal bleaching agents significantly reduced flexural strength (p<0.05. Conclusion: Extracoronal bleaching agents reduce flexural strength, whereas application of CPP-ACP following bleaching either with 15% carbamide peroxide or 40% hydrogen peroxide can increase the flexural strength of enamel-dentin complex.

  5. From selective full-length genes isolation by TAR cloning in yeast to their expression from HAC vectors in human cells.

    Science.gov (United States)

    Kouprina, Natalay; Lee, Nicholas C O; Kononenko, Artem V; Samoshkin, Alexander; Larionov, Vladimir

    2015-01-01

    Transformation-associated recombination (TAR) cloning allows selective isolation of full-length genes and genomic loci as large circular Yeast Artificial Chromosomes (YACs) in yeast. The method has a broad application for structural and functional genomics, long-range haplotyping, characterization of chromosomal rearrangements, and evolutionary studies. In this paper, we describe a basic protocol for gene isolation by TAR as well as a method to convert TAR isolates into Bacterial Artificial Chromosomes (BACs) using a retrofitting vector. The retrofitting vector contains a 3' HPRT-loxP cassette to allow subsequent gene loading into a unique loxP site of the HAC-based (Human Artificial Chromosome) gene delivery vector. The benefit of combining the TAR gene cloning technology with the HAC gene delivery system for gene expression studies is discussed.

  6. Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing E. coli (EHEC and STEC) in cattle.

    Science.gov (United States)

    Dini, C; De Urraza, P J

    2010-09-01

    To isolate, characterize and select phages as potential biocontrol agents of enterohemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) in cattle. Sixteen STEC and EHEC coliphages were isolated from bovine minced meat and stool samples and characterized with respect to their host range against STEC, EHEC and other Gram-negative pathogens; their morphology by electron microscopy; the presence of the stx1, stx2 and cI genes by means of PCR; RAPD and rep-PCR profiles; plaque formation; and acid resistance. Six isolates belonged to the Myoviridae and 10 to the Podoviridae families. The phages negative for stx and cI that formed large, well-defined plaques were all isolated using EHEC O157:H7 as host. Among them, only CA911 was a myophage and, together with CA933P, had the broadest host range for STEC and EHEC; the latter phage also infected Shigella and Pseudomonas. Isolates CA911, MFA933P and MFA45D differed in particle morphology and amplification patterns by RAPD and rep-PCR and showed the highest acidity tolerance. Myophage CA911 and podophages CA933P, MFA933P and MFA45D were chosen as the best candidates for biocontrol of STEC and EHEC in cattle. This work employs steps for a rational selection and characterization of bacteriophages as therapeutic agents. This report constitutes the first documentation of STEC and EHEC phages isolated in Argentina and proposes for the first time the use of rep-PCR as a complement of RAPD on DNA fingerprinting of phages. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  7. Deriving isolates of powdery mildew (Blumeria graminis DC. f.sp. avenae Em. Marchal. in common oat (Avena sativa L. and using them to identify selected resistance genes

    Directory of Open Access Journals (Sweden)

    Sylwia Okoń

    2012-12-01

    Full Text Available Powdery mildew in common oat is caused by Blumeria graminis DC. f.sp. avenae Em. Marchal. Host-pathogen tests are commonly used to identify and locate resistance genes to powdery mildew in cereals. The aim of the study was to determine the virulence of powdery mildew isolates obtained from powdery mildew populations harvested in Poland and to identify OMR1, OMR2 and OMR3 resistance genes to powdery mildew in F2 populations of inter-cultivar hybrids of common oat: Bruno × Fuchs, Jumbo × Fuchs and Mostyn × Fuchs. On the basis of the analysis conducted, isolates enabling division of the studied populations into groups of resistant and susceptible plants were selected. M10 and M14 isolates were chosen for the population which was obtained from crossbreeding of ‘Bruno’ with ‘Fuchs’; these isolates demonstrated avirulence to Bruno cultivar containing OMR1 gene. In order to divide population obtained from crossbreeding of ‘Jumbo’ with ‘Fuchs’, M13 and M16 isolates were chosen; they demonstrated avirulence to the cultivar Jumbo containing the OMR2 gene. On the basis of the tests conducted, it was impossible to select isolates characterised by avirulence to the OMR3 gene. In the F2 population of Bruno × Fuchs and Jumbo × Fuchs hybrids, a division was made into resistant and susceptible plants. The obtained results were verified by the 2 test; the proportion in the dispersion matching model was found to be 3 resistant plants: 1 sensitive plant both in the Bruno × Fuchs and Jumbo × Fuchs populations. Such dispersion indicated that the resistance to powdery mildew in the studied cultivars Bruno and Jumbo was conditioned by single dominant genes.

  8. Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities.

    Science.gov (United States)

    de Los Milagros Orberá Ratón, Teresa; Yano, Ricardo; Rodríguez Gámez, Odalys; Floh, Eny Iochevet Segal; de Jesús Serrat Díaz, Manuel; Barbosa, Heloíza Ramos

    2012-04-01

    Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

  9. Synergistic effects and physiological responses of selected bacterial isolates from animal feed to four natural antimicrobials and two antibiotics.

    Science.gov (United States)

    Zhang, DongLai; Hu, HuiPing; Rao, QinXiong; Zhao, ZhiHui

    2011-10-01

    In this study, 20 samples from three different sources of animal feed were investigated and six bacterial isolates were identified. The susceptibility of four natural antimicrobials, namely, eugenol, cinnamaldehyde, thymol, and carvacrol, against six of these isolates was determined. Carvacrol and eugenol showed better inhibitory effects with larger zones of inhibition. The minimal inhibitory concentration for a range of antibiotics on the susceptibility of two isolates (namely, Sphingomonas paucimobilis and Klebsiella oxytoca) was investigated using the VITEK® 2 microbiological identification system. Both isolates showed a variety of resistance to 18 antibiotics. The minimal inhibitory concentration and fractional inhibitory concentration index of those two isolates for ampicillin and nitrofurantoin in combination with four phenolic compounds was determined. Synergistic interactions were found for most antimicrobial/antibiotic combinations; thymol and carvacrol were very effective (fractional inhibitory concentration ≤0.5) in combination with all antibiotics tested against S. paucimobilis and K. oxytoca, respectively. Ultra performance liquid chromatography techniques were used to investigate the physiological effects of the four natural antimicrobials against those two isolates. Two identical peaks were found to be systematically different between cinnamaldehyde-treated and -untreated cells. The identity of the peaks is unknown and further investigation is needed.

  10. Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics.

    Science.gov (United States)

    Jonas, Kristina; Van Der Vries, Erhard; Nilsson, Mikael T I; Widersten, Mikael

    2005-11-01

    New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

  11. Detection of Extended Spectrum Beta-Lactamases Resistance Genes among Bacteria Isolated from Selected Drinking Water Distribution Channels in Southwestern Nigeria.

    Science.gov (United States)

    Adesoji, Ayodele T; Ogunjobi, Adeniyi A

    2016-01-01

    Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria. In this study, previously described multidrug resistant bacteria from raw, treated, and municipal taps of DWDS from selected dams in southwestern Nigeria were assessed for the presence of ESBL resistance genes which include bla TEM, bla SHV, and bla CTX by PCR amplification. A total of 164 bacteria spread across treated (33), raw (66), and municipal taps (68), belonging to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria group, were selected for this study. Among these bacteria, the most commonly observed resistance was for ampicillin and amoxicillin/clavulanic acid (61 isolates). Sixty-one isolates carried at least one of the targeted ESBL genes with bla TEM being the most abundant (50/61) and bla CTX being detected least (3/61). Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed by Proteus (14.75%). Moreover, combinations of two ESBL genes, bla SHV + bla TEM or bla CTX + bla TEM, were observed in 11 and 1 isolate, respectively. In conclusion, classic bla TEM ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria. These environments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria.

  12. Evaluation and Selection of Multilocus Variable-Number Tandem-Repeat Analysis Primers for Genotyping Brucella abortus Biovar 1 Isolated from Human Patients.

    Science.gov (United States)

    Lee, Subok; Hwang, Kyu-Jam; Park, Mi-Yeoun; Hwang, Seon-Do; Chai, Hee-Youl; Chu, Hyuk; Park, Sang-Hee

    2013-10-01

    Brucellosis is the most common bacterial zoonosis in the world. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is a molecular method for genotyping bacterial species. Brucella abortus biovar I was isolated from most of the brucellosis-suspected patients in Korea. This study was conducted to investigate the ability of various MLVA primers that are used for molecular typing B. abortus isolates and for analyzing their epidemiological data. A total of 80 human isolates of B. abortus biovar I isolated from human patients and the reference strain were used for MLVA. Genetic diversity was determined by calculating the Simpson's diversity index (DI) of each VNTR locus. The Brucella strains were subcultured 30 times to determine the stability of each locus. The DNA of the strains cultivated in each passage was extracted and subjected to MLVA for further investigation. The 15 VNTR loci were selected based on high DI values. The DIs of the 15 VNTR loci showed considerable discrimination power ranging from 59% for Bruce 43 to 87% for Bruce 22. Bruce 09, Bruce 11, Bruce 16, Bruce 42, and Bruce 43 were confirmed to remain stable in vitro among the 15 VNTR loci selected. The results of this study suggest that the five loci subsets may be a useful epidemiological tool for investigating B. abortus biovar 1 outbreak.

  13. Fermentation of Apple Juice with a Selected Yeast Strain Isolated from the Fermented Foods of Himalayan Regions and Its Organoleptic Properties

    OpenAIRE

    Kanwar, S.S.; Keshani,

    2016-01-01

    Twenty-three Saccharomyces cerevisiae strains isolated from different fermented foods of Western Himalayas have been studied for strain level and functional diversity in our department. Among these 23 strains, 10 S. cerevisiae strains on the basis of variation in their brewing traits were selected to study their organoleptic effect at gene level by targeting ATF1 gene, which is responsible for ester synthesis during fermentation. Significant variation was observed in ATF1 gene sequences, sugg...

  14. Evaluation of Two Standard and Two Chromogenic Selective Media for Optimal Growth and Enumeration of Isolates of 16 Unique Bacillus Species.

    Science.gov (United States)

    Kabir, M Shahjahan; Hsieh, Ying-Hsin; Simpson, Steven; Kerdahi, Khalil; Sulaiman, Irshad M

    2017-06-01

    The genus Bacillus is a group of gram-positive endospore-forming bacteria that can cause food poisoning and diarrheal illness in humans. A wide range of food products have been linked to foodborne outbreaks associated with these opportunistic pathogens. The U.S. Food and Drug Administration recommends (in their Bacteriological Analytical Manual) the use of Bacara or mannitol egg yolk polymyxin (MYP) agar plates and the most-probable-number (MPN) method for enumeration and confirmation of Bacillus cereus and related species isolated from foods, sporadic cases, outbreaks, and routine environmental surveillance samples. We performed a comparative analysis of two chromogenic media (Bacara and Brilliance) and two traditional media (MYP and polymyxin egg yolk mannitol bromothymol blue agar [PEMBA]) for the isolation and enumeration of 16 Bacillus species under modified growth conditions that included pH, temperature, and dilution factor. A total of 50 environmental, food, and American Type Culture Collection reference isolates from 16 distinct Bacillus species were evaluated. A food adulteration experiment also was carried out by artificially adulterating two baby food matrices with two isolates each of B. cereus and Bacillus thuringiensis . Our results clearly indicated that chromogenic plating media (Bacara and Brilliance) are better than conventional standard media (MYP and PEMBA) for the detection and enumeration of B. cereus in foods and other official regulatory samples. The comparison of the two chromogenic media also indicated that Brilliance medium to be more efficient and selective for the isolation of Bacillus.

  15. Genotypic Characterization of Azotobacteria Isolated from Argentinean Soils and Plant-Growth-Promoting Traits of Selected Strains with Prospects for Biofertilizer Production

    Directory of Open Access Journals (Sweden)

    Esteban Julián Rubio

    2013-01-01

    Full Text Available The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA, gibberellin (GA3 and zeatin (Z biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2–18.2 μg IAA mL−1, 0.3–0.7 μg GA3 mL−1, and 0.5–1.2 μg Z mL−1. Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

  16. Genotypic characterization of Azotobacteria isolated from Argentinean soils and plant-growth-promoting traits of selected strains with prospects for biofertilizer production.

    Science.gov (United States)

    Rubio, Esteban Julián; Montecchia, Marcela Susana; Tosi, Micaela; Cassán, Fabricio Darío; Perticari, Alejandro; Correa, Olga Susana

    2013-01-01

    The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA), gibberellin (GA3) and zeatin (Z) biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2-18.2 μ g IAA mL(-1), 0.3-0.7 μ g GA3 mL(-1), and 0.5-1.2 μ g Z mL(-1). Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

  17. Pattern of Bacterial Pathogens and Their Susceptibility Isolated from Surgical Site Infections at Selected Referral Hospitals, Addis Ababa, Ethiopia

    Directory of Open Access Journals (Sweden)

    Walelign Dessie

    2016-01-01

    Full Text Available Background. The emergence of multidrug resistant bacterial pathogens in hospitals is becoming a challenge for surgeons to treat hospital acquired infections. Objective. To determine bacterial pathogens and drug susceptibility isolated from surgical site infections at St. Paul Specialized Hospital Millennium Medical College and Yekatit 12 Referral Hospital Medical College, Addis Ababa, Ethiopia. Methods. A cross-sectional study was conducted between October 2013 and March 2014 on 107 surgical site infected patients. Wound specimens were collected using sterile cotton swab and processed as per standard operative procedures in appropriate culture media; and susceptibility testing was done using Kirby-Bauer disc diffusion technique. The data were analyzed by using SPSS version 20. Result. From a total of 107 swabs collected, 90 (84.1% were culture positive and 104 organisms were isolated. E. coli (24 (23.1% was the most common organism isolated followed by multidrug resistant Acinetobacter species (23 (22.1%. More than 58 (75% of the Gram negative isolates showed multiple antibiotic resistance (resistance ≥ 5 drugs. Pan-antibiotic resistance was noted among 8 (34.8% Acinetobacter species and 3 (12.5% E. coli. This calls for abstinence from antibiotic abuse. Conclusion. Gram negative bacteria were the most important isolates accounting for 76 (73.1%. Ampicillin, amoxicillin, penicillin, cephazoline, and tetracycline showed resistance while gentamicin and ciprofloxacin were relatively effective antimicrobials.

  18. Structural Evidence for Direct Interactions Between the BRCT Domains of Human BRCA1 and a Phospho-Peptide from Human ACC1

    Energy Technology Data Exchange (ETDEWEB)

    Shen,Y.; Tong, L.

    2008-01-01

    The tandem BRCA1 C-terminal (BRCT) domains are phospho-serine/threonine recognition modules essential for the function of BRCA1. Recent studies suggest that acetyl-CoA carboxylase 1 (ACC1), an enzyme with crucial roles in de novo fatty acid biosynthesis and lipogenesis and essential for cancer cell survival, may be a novel binding partner for BRCA1, through interactions with its BRCT domains. We report here the crystal structure at 3.2 Angstroms resolution of human BRCA1 BRCT domains in complex with a phospho-peptide from human ACC1 (p-ACC1 peptide, with the sequence 1258-DSPPQ-pS-PTFPEAGH-1271), which provides molecular evidence for direct interactions between BRCA1 and ACC1. The p-ACC1 peptide is bound in an extended conformation, located in a groove between the tandem BRCT domains. There are recognizable and significant structural differences to the binding modes of two other phospho-peptides to these domains, from BACH1 and CtIP, even though they share a conserved pSer-Pro-(Thr/Val)-Phe motif. Our studies establish a framework for understanding the regulation of lipid biosynthesis by BRCA1 through its inhibition of ACC1 activity, which could be a novel tumor suppressor function of BRCA1.

  19. Characterization of gene-environment interactions by behavioral profiling of selectively bred rats: the effect of NMDA receptor inhibition and social isolation.

    Science.gov (United States)

    Petrovszki, Zita; Adam, Gabor; Tuboly, Gabor; Kekesi, Gabriella; Benedek, Gyorgy; Keri, Szabolcs; Horvath, Gyongyi

    2013-03-01

    Gene-environment interactions have an important role in the development of psychiatric disorders. To generate and validate a new substrain of rats with signs related to schizophrenia, we used selective breeding after postweaning social isolation and chronic ketamine treatment through several generations of animals and compared the subsequent strain to naive rats that were not genetically manipulated. We further investigated whether social isolation and ketamine treatment augmented the appearance of schizophrenic-like signs in these rats. Four experimental groups were studied (n=6-15 rats/group): naive rats without any treatment (NaNo); naive rats with postweaning social isolation and ketamine treatment (NaTr); 15th generation of selectively bred animals without any treatment (SelNo) or selectively bred rats with both isolation and ketamine treatment (SelTr). The startle reaction, tail-flick and novel object recognition tests were used to classify the animals into low- or high-risk for schizophrenia. Reduced pain sensitivity, higher degree of the startle reaction, disturbed prepulse inhibition, altered motor activity and decreased differentiation index in the memory test were observed in the 15th generation of the substrain, along with enhanced grooming behavior. Five functional indices (TF latency, startle reaction, prepulse inhibition, differentiation index, and grooming activity) were rated from 0 to 2, and the analysis of the summarized score revealed that the NaNo group had the lowest overall indication of schizophrenic-like signs, while the SelTr animals scored the highest, suggesting that both heritable and environmental factors were important in the generation of the behavioral alterations. We assume that further breeding after this complex treatment may lead to a valid and reliable animal model of schizophrenia. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Natural selection and type 2 diabetes-associated mortality in an isolated indigenous community in the valley of Oaxaca, southern Mexico.

    Science.gov (United States)

    Little, Bertis B; Peña Reyes, Maria Eugenia; Malina, Robert M

    2017-03-01

    This study tests the hypothesis that natural selection is associated with type 2 diabetes (T2D)-associated mortality and fertility in a rural isolated Zapotec community in the Valley of Oaxaca, southern Mexico. Mortality data and related demographic and genealogic information were linked with data for fertility, prereproductive mortality and family history of mortality attributed to T2D. Physician verified T2D mortality (n = 27) between 1980 and 2009 and imputed T2D (n = 70) from cardiovascular mortality (68% random sample) and renal failure (44% random sample). Bootstrapping was used to obtain a robust variance estimate in survival analysis and multivariate analysis of variance. Estimated maximum natural selection by Crow's Index occurred circa 1930 and was relaxed after this time in the study population. Cox-regression survival analysis of T2D mortality with covariates (family history of T2D, cardiovascular disease, renal failure) indicated a significant hazard ratio (HR = 5.95, 95% CI: 1.38-25.67, p natural selection decreased, and favored offspring survival of non-T2D descedants. The results indicated statistically significant directional selection against T2D and imputed T2D to this population isolate. © 2016 Wiley Periodicals, Inc.

  1. Spatially heterogeneous environmental selection strengthens evolution of reproductively isolated populations in a Dobzhansky-Muller system of hybrid incompatibility

    Science.gov (United States)

    Samuel A. Cushman; Erin L. Landguth

    2016-01-01

    Within-species hybrid incompatibility can arise when combinations of alleles at more than one locus have low fitness but where possession of one of those alleles has little or no fitness consequence for the carriers. Limited dispersal with small numbers of mate potentials alone can lead to the evolution of clusters of reproductively isolated genotypes despite...

  2. Evaluations and modifications of semi-selective media for improved isolation of Agrobacterium tumefaciens biovar 1 from cultivated walnut

    Science.gov (United States)

    Agrobacterium tumefaciens, the causal agent of crown gall of walnut, is an aerobic, Gram negative bacterium belonging to the family Rhizobiaceae. Like many in this group, A. tumefaciens is a common inhabitant of soil and plant host tissue. Isolation from these complex environments is difficult even ...

  3. VapB type 8 plasmids in Rhodococcus equi isolated from the small intestine of pigs and comparison of selective culture media.

    Science.gov (United States)

    Lara, G H B; Takai, S; Sasaki, Y; Kakuda, T; Listoni, F J P; Risseti, R M; de Morais, A B C; Ribeiro, M G

    2015-09-01

    The virulence-plasmid profile of Rhodococcus equi strains isolated from Suidae and humans is similar. Recent evidence suggests that the consumption of pork products contaminated with faeces might be a potential source of R. equi infections in humans, mainly to patients with rhodococcosis without history of contact with pigs or pig farms. This study investigated the virulence-associated genes (vapA and vapB) and plasmid profiles of R. equi among the 150 samples of small intestinal content obtained from slaughtered pigs. In addition, all samples were subjected to microbiological culture in conventional sheep blood agar and CAZ-NB, TCP and TVP selective media. A total of 40 (26·7%) of the samples recovered R. equi, with two samples recovering isolates harbouring the VapB type 8 plasmid. Among the 150 pigs sampled herein, CAZ-NB was considered the best selective medium for the isolation of R. equi from faeces. Our results provide evidence that the contamination of slaughtered pig carcasses with pathogenic R. equi might occur through faeces, representing a public health concern. Furthermore, this study is the first description of R. equi strains carrying the VapB plasmid in the gut of pigs. Intermediately virulent (VapB) is a common plasmid-type harboured by R. equi isolated from pigs and humans with AIDS. Curiously, humans with rhodococcosis usually have no history of contact with pigs or pig farms. Virulence-plasmid profile of 40 R. equi isolated among 150 small intestine content samples from pigs revelled two carrying isolates with the VapB type-8 plasmids. Moreover, comparison of three selective culture media shows that CAZ-NB was the best. Our results provide evidence that contamination of slaughtered pig carcasses with pathogenic R. equi might occur through faeces, representing a public health concern. Furthermore, R. equi carrying VapB type-8 plasmids types are described for the first time in the gut of the pig. © 2015 The Society for Applied

  4. Directed analysis of cyanobacterial membrane phosphoproteome using stained phosphoproteins and titanium-enriched phosphopeptides.

    Science.gov (United States)

    Lee, Dong-Gi; Kwon, Joseph; Eom, Chi-Yong; Kang, Young-Moon; Roh, Seong Woon; Lee, Kyung-Bok; Choi, Jong-Soon

    2015-04-01

    Gel-free shotgun phosphoproteomics of unicellular cyanobacterium Synechocystis sp. PCC 6803 has not been reported up to now. The purpose of this study is to develop directed membrane phosphoproteomic method in Synechocystis sp. Total Synechocystis membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphoprotein-stained gel bands were selectively subjected to in-gel trypsin digestion. The phosphorylation sites of the resulting peptides were determined by assigning the neutral loss of [M-H(3)PO(4)] to Ser, Thr, and Tyr residues using nano-liquid chromatography 7 Tesla Fourier transform mass spectrometry. As an initial application, 111 proteins and 33 phosphoproteins were identified containing 11 integral membrane proteins. Identified four unknown phosphoproteins with transmembrane helices were suggested to be involved in membrane migration or transporters based on BLASTP search annotations. The overall distribution of hydrophobic amino acids in pTyr was lower in frequency than that of pSer or pThr. Positively charged amino acids were abundantly revealed in the surrounding amino acids centered on pTyr. A directed shotgun membrane phosphoproteomic strategy provided insight into understanding the fundamental regulatory processes underlying Ser, Thr, and Tyr phosphorylation in multi-layered membranous cyanobacteria.

  5. Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

    NARCIS (Netherlands)

    Butré, C.I.; Sforza, S.; Gruppen, H.; Wierenga, P.A.

    2014-01-01

    Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each

  6. Conceptual design and selection of deposited metal on the valve seat for the high temperature isolation valve in the HTTR hydrogen production system (contract research)

    Energy Technology Data Exchange (ETDEWEB)

    Nishihara, Tetsuo; Iwatsuki, Jin [Department of Advanced Nuclear Heat Technology, Oarai Research Establishment, Japan Atomic Energy Research Institute, Oarai, Ibaraki (Japan)

    1999-11-01

    In the HTTR hydrogen production system a high temperature isolation valve should be provided outside the penetration of containment vessel on the secondary helium loop. As the secondary helium gas is at temperature of 905degC and pressure of 4.1 MPa, there is not any available isolation valve used under such conditions. Once demonstration test of high temperature isolation valve was carried out in the ERANS project. Tested valve could meet the basic design requirements but some problems remained. In this report a conceptual design of the high temperature isolation valve is performed in consideration of resolving these problems. The structural reliability is confirmed by the three-dimensional stress analysis. With respect to the deposited metal on valve seat, a screening test is done to observe the basic properties of candidate metals. From these results, it is shown that Stellite alloy that is used in practical valve has a possibility of the separation at welding layer and carbon diffusion from deposited metal into the base metal. Nickel-base super alloy has a possibility of internal and intergranular oxidation due to contained Al and Ti. Therefore, detail metallographical and mechanical tests for long period are planned to select the adequate deposited metal. (author)

  7. First Direct Isolation of Stable α-Form Crystals of Mirabegron, a Selective β3-Adrenoceptor Agonist.

    Science.gov (United States)

    Deshmukh, G Dattatray; Bangal, N Mukund; Mali, C Anil; Medhane, J Vijay; Mathad, T Vijayavitthal

    2017-06-01

    An efficient and scalable method for the direct isolation of stable α-form crystals of Mirabegron (1) is developed. The developed method negates transformation of metastable β-form crystals into α-form crystals thereby overcoming the limitations of reported methods and avoids additional processing steps during its manufacture. The developed method directly provides stable α-form crystals of Mirabegron (1) with yield of around 84% and purity of >99.77% by HPLC in a single step.

  8. The effects of sodium hypochlorite against selected drinking water-isolated bacteria in planktonic and sessile states

    OpenAIRE

    Gomes, I. B.; Simões, M.; Simões, Lúcia C.

    2016-01-01

    Chlorine is the most commonly used agent for general disinfection, particularly for microbial growth control in drinking water distribution systems. The goals of this study were to understand the effects of chlorine, as sodium hypochlorite (NaOCl), on bacterial membrane physicochemical properties (surface charge, surface tension and hydrophobicity) and on motility of two emerging pathogens isolated from drinking water, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. The effects ...

  9. Isolation of the four methyl jasmonate stereoisomers and their effects on selected chiral volatile compounds in red raspberries.

    Science.gov (United States)

    Flores, Gema; Blanch, Gracia Patricia; Ruiz del Castillo, Maria Luisa

    2013-12-01

    The four stereoisomers present in a commercial sample of methyl jasmonate (MJ) were isolated at semi-preparative scale by HPLC, using a permethylated β-cyclodextrin column. This allowed the baseline resolution and collection of both major (methyl jasmonates) and minor (epi-methyl jasmonates) stereoisomers. When 1.5 mL of a 5mg per mL MJ solution were injected, isolated amounts were 3.56 mg for (-) and (+)-methyl jasmonates, with respective purities of 96.1% and 99.9%, and 0.18 mg for (-)- and (+)-epi-methyl jasmonates, with 98.6% and 91.6% respective purities. The post-harvest treatment of red raspberry fruits with the pure methyl jasmonate stereoisomers isolated proved that (-)-epi-MJ either promotes the bioformation of (+)-limonene or inhibits that of (-)-limonene to a greater extent than the other three MJ stereoisomers, while the biosynthesis of the (+)-enantiomer of α-ionone is favoured equally, whichever MJ stereoisomer used. The results obtained in the present study might be used to obtain food products with improved sensory characteristics. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Isolation of potentially pathogenic fungi from selected pigeons' feeding sites in Karachi: A new dimension to health hazard.

    Science.gov (United States)

    Naz, Sehar Afshan; Yaseen, Muhammad; Jabeen, Nusrat; Shafique, Maryam

    2017-06-01

    To determine the presence of pathogenic fungal strains in areas where pigeons are present in a large number. This study was conducted at the Federal Urdu University of Arts, Science and Technology, Karachi, from February 2015 to March2016, and comprised samples of soil contaminated with pigeons' excreta. The samples were collected from 20 different pigeon-feeding places in the city. These samples were processed for the isolation and identification of fungi by using standard conventional methods. The fungal strains isolated were also tested for their susceptibility to commonly used antifungal agents by disc diffusion technique. There were 105 samples. A wide variety of fungal strains belonging to different genera of Aspergillus, Rhizopus, Penicillium, Fusarium and Candida were isolated and identified by using conventional methods. The antifungal resistance pattern of these strains also depicts emergence of resistance against commonly used antifungal agents such as amphotericin B and fluconazole. The soil and air of places densely populated with pigeons were found to be loaded with fungal spores and many of them were potential pathogens.

  11. Prevalence, antimicrobial resistance profile and comparison of selective plating media for the isolation of Salmonella in backyard chickens from Entre Rios, Argentina.

    Science.gov (United States)

    Rodríguez, F I; Pascal, D C; Pulido, D; Osinalde, J M; Caffer, M I; Bueno, D J

    2017-10-27

    This study was conducted to estimate the apparent prevalence of Salmonella spp. in birds kept under backyard system in Entre Ríos, Argentina, and determine the performance of two selective plating media used for Salmonella isolation, and the antimicrobial resistance of the isolated. Also, the association of farms characteristics with Salmonella presence was evaluated. A total of 657 backyard chickens and 15 gooses were sampled one time by cloacal swab, belonging to 51 and one family farms, respectively, and four counties in Entre Rios state from April 2014 to May 2015. Only four samples from backyard chickens belonged to three family farms from Uruguay County were positive to Salmonella spp., so the apparent prevalence was 0.6% for this kind of chicken. Four serovars were isolated (Salmonella ser. Lille, S. ser. Newport, S. ser. Enteritidis and S. ser. Rissen), which were susceptible to all antibiotics tested with the exception of erythromycin. For Hektoen enteric agar and brilliant green agar, relative specificity and positive predictive value were 1, and the relative sensitivity and negative predictive value did not show any difference between them. The agreement was very good between these two plating media. None of the variables studied could be selected to calculate the risk factors associated with Salmonella isolation because p > .15. Although the prevalence of Salmonella spp. is low in backyard birds in Entre Rios, the presence of S. ser. Enteritidis should not be discounted, because it is found in the county that concentrates a large population of intensive poultry production in the state. © 2017 Blackwell Verlag GmbH.

  12. The Polymyxin Ceftazidime Oxford Medium as an alternative selective and differential medium for isolation of Listeria monocytogenes from raw or unpasteurized food.

    Science.gov (United States)

    Martínez-Gonzáles, N E; Martínez-Chávez, L; Martínez-Cárdenas, C; Cabrera-Díaz, E; Castillo, A

    2014-04-01

    The Polymyxin Ceftazidime Oxford Medium (PCOM) was developed to recover Listeria monocytogenes from raw or unpasteurized foods. It contains esculin-ferric ammonium citrate as indicator system for Listeria growth, and ceftazidime and polymyxin B as selective agents, which are available in several Latin American countries. Comparison of PCOM, Modified Oxford Medium (MOX) and Tryptic Soy agar with 0.6% yeast extract (TSAYE) indicated that both selective media were equally effective at recovering four individual strains of L. monocytogenes (Scott A, V7, California and broccoli), and a mixture of these strains (LMM) (P > 0.05). The ability of PCOM, MOX, TSAYE and TSAYE supplemented with 4% NaCl to recover heat, acid and freeze-damaged LMM was similar for all media (P > 0.05). The PCOM proved to be effective at isolating colonies of LMM from inoculated raw beef chunks, unpasteurized orange juice, cabbage, and Mexican-style cheese by direct plating and by the US Department of Agriculture's Food Safety and Inspection Service enrichment method. Differentiation of L. monocytogenes colonies was easier on PCOM than on MOX for foods with high levels of background microbiota. Based on the evaluations performed on foods naturally contaminated with L. monocytogenes, PCOM was a more economical alternative than MOX for selective and differential isolation of Listeria from raw or unpasteurized foods. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Elgersma, Y.; van den Berg, M.; Tabak, H. F.; Distel, B.

    1993-01-01

    To study peroxisome biogenesis, we developed a procedure to select for Saccharomyces cerevisiae mutants defective in peroxisomal protein import or peroxisome assembly. For this purpose, a chimeric gene was constructed encoding the bleomycin resistance protein linked to the peroxisomal protein

  14. Prevalence of Pathogenic Bacteria Isolated from Surgical Site and Wound Infection among Patients Admitted in some selected Hospitals in Sokoto Metropolis, Nigeria

    Directory of Open Access Journals (Sweden)

    UK Muhammad

    2014-09-01

    Full Text Available Surgical and open wounds are commonly encountered in clinical practice. This study was aim to determine the prevalence of pathogenic bacteria in surgical and open wound infection among patients admitted in some selected hospitals in Sokoto metropolis. A total of one hundred and fifty one (151 isolates were obtained from two hundred (200 surgical site and wound samples collected from patients in this study. The result showed that Usmanu Danfodiyo Teaching Hospital Sokoto (UDUTH had the highest number of clinical isolates with 64 gram positive and gram negative bacteria followed by Specialist Hospital Sokoto (S.H.S with 57 gram positive and gram negative bacteria and then Maryam Abacha Women and Children Hospital (MAWCH with 30 gram positive and gram negative bacteria. Gram positive cocci 108 (71.5% were more predominant pathogen isolated in the hospitals than gram negative bacilli 43 (28.5%. Staphylococcus aureus had the highest number of occurrence with 54(35.76% followed by Coagulate negative Staphylococci with 47(31.1% while Citrobacter freundii had the lowest number of occurrence with 2(1.32% isolates. Also, the susceptibility of the isolates to antimicrobial agents were carried out using Amoxacillin, Ampicillin, Erythromycin, Chloramphenicol, Ampiclox, Ciprofloxacin, Gentamycin, Tetracycline, Pefloxacin and Cotrimoxazole. The mean zone of inhibition recorded against Staphlococcus aureus by using Amoxacillin antibiotic is 2.20mm while with Citrobacter freundii is 1.00. DOI: http://dx.doi.org/10.3126/ije.v3i3.11066 International Journal of Environment Vol.3(3 2014: 89-103

  15. Preliminary selection for potential probiotic Bifidobacterium isolated from subjects of different Chinese ethnic groups and evaluation of their fermentation and storage characteristics in bovine milk.

    Science.gov (United States)

    Liu, W J; Chen, Y F; Kwok, L Y; Li, M H; Sun, T; Sun, C L; Wang, X N; Dan, T; Menghebilige; Zhang, H P; Sun, T S

    2013-01-01

    A total of 29 strains of Bifidobacterium were isolated from 18 samples of human feces in different ethnic minority regions of China. All isolates were identified as Bifidobacterium longum (9 strains) and Bifidobacterium pseudocatenulatum (20 strains) based on 16S rRNA gene sequencing and phylogenetic analysis. These strains were preliminarily tested for their suitability to become probiotics by assessing their ability to survive adequately at low pH conditions and their tolerance of different concentrations of bile salts and simulated gastrointestinal juices. In vitro tests were sequentially used to predict the survival of these strains in the simulated conditions in the human gastrointestinal tract. These strains were first exposed to pH 2.5 for 3h, and 7 out of the 29 strains were discriminated from the others by their high survival rates. Out of these 7 strains, 4 were found to grow and survive well at an even lower pH of 2.0 and in high bile salt concentration. Apart from the gastrointestinal survival capacity, both fermentation efficiency and storage characteristics are important criteria for selecting for suitable potential probiotic strains. Therefore, the fermentation efficiency in bovine milk and the bacterial viability during the storage in the resultant fermented milk were also evaluated for these 4 selected strains. In this study, we isolated and identified 29 novel Bifidobacterium strains. Based on our initial evaluation, at least 4 of them may serve as valuable resources for further dairy probiotic strain selection. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  16. Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

    OpenAIRE

    Thalyta Marina Benetti; Cristina Leise Bastos Monteiro; Márcia Regina Beux; Wanda Moscalewski Abrahão

    2012-01-01

    The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars) with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA) and CM 1084). The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite ...

  17. Selection of potential Enterococcus faecium isolated from Thai native chicken for probiotic use according to the in vitro properties

    Directory of Open Access Journals (Sweden)

    Napaporn Lertworapreecha

    2011-02-01

    Full Text Available Sixty strains of E. faecium were isolated from 30 samples of native chickens’ gastrointestinal tracts. All strains weretested on acid and bile tolerance. Fifteen strains passed the acid tolerance test. The best five strains were EFMC 17, 21 and24; EFMD 25; EFMI 47 and 49. Only four strains, EFMC 21; EFMD 30; EFMI 47, and 49, survived 4 hours of bile exposure.Fifteen strains that passed the acid tolerance test were tested for their ability of intestinal mucus attachment. The resultsindicated that all strains were able to attach to intestinal mucus. For the ability of pathogenic bacteria inhibition test, theresult found seven strains (EFMC 17, 21 and 24; EFMD 29 and 30; EFMI 46 and 49 showed better performance than strainEFC. All seven strains were acid producer, but only four strains (EFMC 21; EFMD 25; EFMI 47 and 49 were able to releasebacteriocin. Based on proper probiotic properties two strains (EFMI 47 and 49 of E. faecium isolated from Thai native chicksin this study have a potential use as probiotics. Antimicrobial susceptibility test of these two strains have been also performed;they were susceptible to amoxicillin/clavulanic, ciprofloxacin, gentamycin, trimethoprime/sulphamethoxazole, vancomycin,and trimethoprim. On the other hand, they were resistant to cefotaxime, erythromycin, and tetracycline. The DNA-DNAhybridization percentage of DNA-DNA homology to E. faecium NRIC 1145 of EFMI 47 and EFMI 49 were 82.36 and 78.63%,respectively.

  18. Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies.

    Science.gov (United States)

    Su, Yu-Cheng; Al-Qaisi, Talal S; Tung, Hsin-Yi; Cheng, Tian-Lu; Chuang, Kuo-Hsiang; Chen, Bing-Mae; Roffler, Steve R

    2014-01-01

    Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative "cloning-free" approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.

  19. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

    Science.gov (United States)

    Tullila, Antti; Nevanen, Tarja K.

    2017-01-01

    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  20. Synergistic Effects of n-Hexane Fraction of Parkia biglobosa (Jacq. Bark Extract and Selected Antibiotics on Bacterial Isolates

    Directory of Open Access Journals (Sweden)

    Oluwatayo E. Abioye

    2017-02-01

    Full Text Available The incidence of resistance to commonly used antimicrobial agents by microbial pathogens demands increased effort in the development of effective ways of treating infections and diseases. The n-hexane fraction of lyophilized crude bark extract of Parkia biglobosa (Jacq. was prepared and, in combination with selected antibiotics, assayed for antimicrobial activity against some selected bacterial pathogens using time-kill assay. Protein leakage analysis of the combined agents was performed using Bradford protein quantification method. Determination of active compounds present in the n-hexane fraction was done using Fourier Transform Infrared Spectroscopy (FTIR. While time-kill assay detected 43.33% synergy; 56.67% indifference and no antagonism at 1/2 × minimum inhibitory concentration (MIC, 1 × MIC exhibited 55% synergy, 45% indifference and no antagonism. Protein leakages from the cells of selected bacteria ranged from 1.20 µg/mL to 256.93 µg/mL. The presence of a phenyl group, an aromatic ring and phenolic compounds in the n-hexane fraction was confirmed at 2162 cm−1–2020 cm−1, 1605 cm−1–1533 cm−1 and 1438 cm−1–1444 cm−1 spectra peaks, respectively. The observed antibiotic−n-hexane fraction synergistic interaction revealed the improved antibacterial activity of the selected antibiotics. Hence, exploration of a combination of antibiotics with plant secondary metabolites is hereby advocated in the global quest for means of combating infectious diseases caused by multidrug-resistant pathogens.

  1. Selective injection and isolation of ions in quadrupole ion trap mass spectrometry using notched waveforms created using the inverse Fourier transform

    Energy Technology Data Exchange (ETDEWEB)

    Soni, M.H.; Cooks, R.G. (Purdue Univ., West Lafayette, IN (United States))

    1994-08-01

    Broad-band excitation of ions is accomplished in the quadrupole ion trap mass spectrometer using notched waveforms created by the SWIFT (stored waveform inverse Fourier transform) technique. A series of notched SWIFT pulses are applied during the period of ion injection from an external Cs[sup +] source to resonantly eject all ions whose resonance frequencies fall within the frequency range of the pulse while injecting only those analyte ions whose resonance frequencies fall within the limits of the notch. This allows selective injection and accumulation of the ions of interest and continuous ejection of the unwanted ions. This is shown to result in significant improvement in S/N ratio, resolution, and sensitivity for the analyte ions of interest. Selective ion injection is demonstrated by injecting the protonated molecules of peptides VSV and gramicidin S and the intact cation of l-carnitine hydrochloride, using singly notched SWIFT pulses. Multiply notched SWIFT pulses are used to simultaneously inject ions of different m/z values of l-carnitine hydrochloride into the ion trap. A new coarse/fine ion isolation procedure, which employs a doubly notched SWIFT pulse, is demonstrated for isolating ions of a single m/z value of 4-bromobiphenyl from a population of trapped ions. 36 refs., 10 figs., 2 tabs.

  2. Evaluation of phenylthiocarbamoyl-derivatized peptides by electrospray ionization mass spectrometry: selective isolation and analysis of modified multiply charged peptides for liquid chromatography-tandem mass spectrometry experiments.

    Science.gov (United States)

    Sanchez, Aniel; Perez-Riverol, Yasset; González, Luis Javier; Noda, Jesus; Betancourt, Lazaro; Ramos, Yassel; Gil, Jeovanis; Vera, Roberto; Padrón, Gabriel; Besada, Vladimir

    2010-10-15

    Edman degradation in the gas phase has been observed by collision activated dissociation of N-terminal phenylthiocarbamoyl (PTC) protonated peptide to yield abundant complementary b₁ and y(n-1) ion pairs. Here, we demonstrated the relation between the observed losses of aniline and/or the entire PTC derivatizing group with the availability of mobile protons using electrospray ionization mass spectrometry. In order to select the peptides with more efficient fragmentation, while simplifying the mixture of peptides, we extend the phenylisotiocyanate (PITC) derivatization of amino groups to the selective isolation of multiply charged peptides (those having the number of arginines and histidines residues higher than one) using a procedure previously developed in our group. Thus, it was possible to identify in the filtered protein database the sequence of the isolated multiply charged peptides derived from a single protein and a complex mixture of proteins extracted from Escherichia coli using only the molecular mass and the N-terminal amino acid information. For this purpose, we developed a novel bioinformatic tool for automatic identification of peptides from liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments, which potentially can be used in high-throughput proteomics.

  3. Computational analysis of phosphopeptide binding to the polo-box domain of the mitotic kinase PLK1 using molecular dynamics simulation.

    Directory of Open Access Journals (Sweden)

    David J Huggins

    2010-08-01

    Full Text Available The Polo-Like Kinase 1 (PLK1 acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD. The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.

  4. In vitro pre-selection criteria for probiotic Lactobacillus acidophilus TS1 isolated from fermented milk product, Dahi

    Directory of Open Access Journals (Sweden)

    Maqsood, S.

    2013-12-01

    Full Text Available Aims: The aim of this research was to evaluate some probiotic traits of Lactobacillus acidophilus TS1 strain previously isolated from dahi. Methodology and results: For this purpose, tested strain was evaluated for their resistance to low pH, tolerance to bile and in vitro antibiotics susceptibility. It was observed that the strain TS1 remained viable at pH 3.0 to 6 and bile concentration of 0.1 to 0.3%. Conclusion, significance and impact of study: L. acidophilus TS1 was found resistant to nalidixic acid, ciprofloxacine, gentamycin and vancomycin. The results highlighted the probiotic potential of L. acidophilus TS1 which deserves further investigation in vitro studies to elucidate its health benefits.

  5. Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths.

    Science.gov (United States)

    Gorski, Lisa

    2012-01-01

    For foodborne outbreak investigations it can be difficult to isolate the relevant strain from food and/or environmental sources. If the sample is contaminated by more than one strain of the pathogen the relevant strain might be missed. In this study mixed cultures of Salmonella enterica were grown in one set of standard enrichment media to see if culture bias patterns emerged. Nineteen strains representing four serogroups and ten serotypes were compared in four-strain mixtures in Salmonella-only and in cattle fecal culture enrichment backgrounds using Salmonella enrichment media. One or more strain(s) emerged as dominant in each mixture. No serotype was most fit, but strains of serogroups C2 and E were more likely to dominate enrichment culture mixtures than strains of serogroups B or C1. Different versions of Rappaport-Vassiliadis (RV) medium gave different patterns of strain dominance in both Salmonella-only and fecal enrichment culture backgrounds. The fittest strains belonged to serogroups C1, C2, and E, and included strains of S. Infantis, S. Thompson S. Newport, S. 6,8:d:-, and S. Give. Strains of serogroup B, which included serotypes often seen in outbreaks such as S. Typhimurium, S. Saintpaul, and S. Schwarzengrund were less likely to emerge as dominant strains in the mixtures when using standard RV as part of the enrichment. Using a more nutrient-rich version of RV as part of the protocol led to a different pattern of strains emerging, however some were still present in very low numbers in the resulting population. These results indicate that outbreak investigations of food and/or other environmental samples should include multiple enrichment protocols to ensure isolation of target strains of Salmonella.

  6. Selected Essential Oils as Antifungal Agents Against Antibiotic-Resistant Candida spp.: In Vitro Study on Clinical and Food-Borne Isolates.

    Science.gov (United States)

    Rajkowska, Katarzyna; Kunicka-Styczyńska, Alina; Maroszyńska, Marta

    2017-01-01

    Candida spp. cause significant health problems, inducing various types of superficial and deep-seated mycoses in humans. As a result of the increasing antibiotic resistance among pathogenic yeasts, the interest in alternative agents of antifungal activity is growing. This study evaluated the antimicrobial activity of selected essential oils (EOs) against Candida clinical and food-borne strains, including antibiotic-resistant isolates, in relation to yeast cell surface hydrophobicity (CSH). Candida strains showed different range of susceptibility to tea tree, thyme, peppermint, and clove oils, and peppermint oil demonstrated the lowest anticandidal activity with minimal inhibitory concentrations (MICs) of 0.03-8.0% v/v. MIC values for thyme and clove oils ranged from 0.03% to 0.25% v/v, and for tea tree oil-from 0.12% to 2.0% v/v. The exception was Candida tropicalis food-borne strain, the growth of which was inhibited after application of EOs at concentration of 8% v/v. Due to diverse yeast susceptibility to EOs, isolates were divided into five clusters in a principal component analysis model, each containing both clinical and food-borne strains. Hydrophobic properties of yeast were also diversified, and 37% of clinical and 50% of food-borne strains exhibited high hydrophobicity. The study indicates high homology of clinical and food-borne Candida isolates in relation to their susceptibility to anticandidal agents and hydrophobic properties. The susceptibility of yeasts to EOs could be partially related to their CSH. High antifungal activity of examined EOs, also against antibiotic-resistant isolates, indicates their usefulness as agents preventing the development of Candida strains of different origin.

  7. Synthesis and functionalization of superparamagnetic poly-ɛ-caprolactone microparticles for the selective isolation of subpopulations of human adipose-derived stem cells

    Science.gov (United States)

    Balmayor, Elizabeth R.; Pashkuleva, Iva; Frias, Ana M.; Azevedo, Helena S.; Reis, Rui L.

    2011-01-01

    There has been a growing interest in using biofunctionalized magnetic particles for cell isolation. This paper describes the synthesis and characterization of magnetite-polymer (Fe3O4-poly-ɛ-caprolactone, magnetite-PCL) microparticles surface functionalized with amino and epoxy groups allowing easy covalent attachment of specific antibodies and subsequent ability to bind target cells. Particles with different sizes (4–135 µm), spherical shape and superparamagnetic behaviour (magnetite content of about 13 wt%) were obtained. The functionalized microparticles presented high protein-binding capacity (coupling efficiency of 47% for epoxy- and 71% for amino-functionalized particles) with a low level of non-specific binding. We have further investigated the influence of initial protein concentration, pH, ionic strength, temperature and incubation time on the capacity of amino-functionalized particles to bind protein molecules. The results showed that maximum protein coupling is rapidly achieved (≤5 h) at pH 5.5 and low ionic strength (0.05 M NaCl). Furthermore, when cultured in direct contact with osteoblast-like cells (Saos-2) or human-derived adipose stem cells (ASCs), the amino-functionalized particles did not affect the proliferation and morphology of the cells. As a proof of principle for the application of magnetic microparticles for cell isolation, CD105 (endoglin) antibody was coupled to the magnetic particle surface to bind subpopulations of human ASCs expressing the CD105 antigen. The isolation of CD105+ ASCs from a heterogeneous cell population was confirmed by flow cytometry analysis. Given the demonstrated potential of functionalized magnetite-PCL microparticles for selective cell isolation, we expect that these particles may be further applied in immuno-magnetic cell separation owing to their versatility and ease of surface modification. PMID:21208971

  8. Repeated Aspergillus isolation in respiratory samples from non-immunocompromised patients not selected based on clinical diagnoses: colonisation or infection?

    Science.gov (United States)

    Barberan, Jose; Alcazar, Bernardino; Malmierca, Eduardo; Garcia de la Llana, Francisco; Dorca, Jordi; Del Castillo, Daniel; Villena, Victoria; Hernandez-Febles, Melissa; Garcia-Perez, Francisco-Javier; Granizo, Juan-Jose; Gimenez, Maria-Jose; Aguilar, Lorenzo

    2012-11-12

    Isolation of Aspergillus from lower respiratory samples is associated with colonisation in high percentage of cases, making it of unclear significance. This study explored factors associated with diagnosis (infection vs. colonisation), treatment (administration or not of antifungals) and prognosis (mortality) in non-transplant/non-neutropenic patients showing repeated isolation of Aspergillus from lower respiratory samples. Records of adult patients (29 Spanish hospitals) presenting ≥ 2 respiratory cultures yielding Aspergillus were retrospectively reviewed and categorised as proven (histopathological confirmation) or probable aspergillosis (new respiratory signs/symptoms with suggestive chest imaging) or colonisation (symptoms not attributable to Aspergillus without dyspnoea exacerbation, bronchospasm or new infiltrates). Logistic regression models (step-wise) were performed using Aspergillosis (probable + proven), antifungal treatment and mortality as dependent variables. Significant (p < 0.001) models showing the highest R2 were considered. A total of 245 patients were identified, 139 (56.7%) with Aspergillosis. Aspergillosis was associated (R2 = 0.291) with ICU admission (OR = 2.82), congestive heart failure (OR = 2.39) and steroids pre-admission (OR = 2.19) as well as with cavitations in X-ray/CT scan (OR = 10.68), radiological worsening (OR = 5.22) and COPD exacerbations/need for O2 interaction (OR = 3.52). Antifungals were administered to 79.1% patients with Aspergillosis (100% proven, 76.8% probable) and 29.2% colonised, with 69.5% patients receiving voriconazole alone or in combination. In colonised patients, administration of antifungals was associated with ICU admission at hospitalisation (OR = 12.38). In Aspergillosis patients its administration was positively associated (R(2) = 0.312) with bronchospasm (OR = 9.21) and days in ICU (OR = 1.82) and negatively with Gold III + IV (OR = 0.26), stroke (OR = 0.024) and quinolone treatment (OR = 0

  9. Actinobacteria phylogenomics, selective isolation from an iron oligotrophic environment and siderophore functional characterization, unveil new desferrioxamine traits.

    Science.gov (United States)

    Cruz-Morales, Pablo; Ramos-Aboites, Hilda E; Licona-Cassani, Cuauhtémoc; Selem-Mójica, Nelly; Mejía-Ponce, Paulina M; Souza-Saldívar, Valeria; Barona-Gómez, Francisco

    2017-09-01

    Desferrioxamines are hydroxamate siderophores widely conserved in both aquatic and soil-dwelling Actinobacteria. While the genetic and enzymatic bases of siderophore biosynthesis and their transport in model families of this phylum are well understood, evolutionary studies are lacking. Here, we perform a comprehensive desferrioxamine-centric (des genes) phylogenomic analysis, which includes the genomes of six novel strains isolated from an iron and phosphorous depleted oasis in the Chihuahuan desert of Mexico. Our analyses reveal previously unnoticed desferrioxamine evolutionary patterns, involving both biosynthetic and transport genes, likely to be related to desferrioxamines chemical diversity. The identified patterns were used to postulate experimentally testable hypotheses after phenotypic characterization, including profiling of siderophores production and growth stimulation of co-cultures under iron deficiency. Based in our results, we propose a novel des gene, which we term desG, as responsible for incorporation of phenylacetyl moieties during biosynthesis of previously reported arylated desferrioxamines. Moreover, a genomic-based classification of the siderophore-binding proteins responsible for specific and generalist siderophore assimilation is postulated. This report provides a much-needed evolutionary framework, with specific insights supported by experimental data, to direct the future ecological and functional analysis of desferrioxamines in the environment. © FEMS 2017.

  10. Combination Effect of Antituberculosis Drugs and Ethanolic Extract of Selected Medicinal Plants against Multi-Drug Resistant Mycobacterium tuberculosis Isolates

    Directory of Open Access Journals (Sweden)

    Prabasiwi Nur Fauziyah

    2017-03-01

    Full Text Available Adverse drug reaction and resistance to antituberculosis drugs remain the causes of tuberculosis therapeutic failure. This research aimed to find the combination effect of standard antituberculosis drugs with Hibiscus sabdariffa L., Kaempferia galanga L., and Piper crocatum N.E. Br against multi-drug resistant (MDR Mycobacterium tuberculosis isolates. Two MDR strains (i.e., isoniazid/ethambutol resistant and rifampicin/streptomycin resistant of M. tuberculosis were inoculated in Löwenstein–Jensen medium containing a combination of standard antituberculosis drugs and ethanolic extracts of H. sabdariffa calyces, K. galanga rhizomes, and P. crocatum leaves using various concentration combinations of drug and extract. The colony numbers were observed for 8 weeks. The effect of the combination was analyzed using the proportion method which was calculated by the mean percentage of inhibition reduction in a number of colonies on drug–extract containing medium compared to extract-free control medium. The results showed that all three plant extracts achieved good combination effects with rifampicin against the rifampicin/streptomycin resistant strain. Antagonistic effects were, however, observed with streptomycin, ethambutol and isoniazid, therefore calling for caution when using these plants in combination with antituberculosis treatment.

  11. Phytochemical and antibacterial activity of the extracts of Fagara zanthoxyloides on selected cariogenic and enteric bacterial isolates

    Directory of Open Access Journals (Sweden)

    Martins Ajibade Adefisoye

    2012-02-01

    Methods: Crude extracts were obtained by cold extraction method of the powdered stem in methanol-water mixture (MW in ratio 3:2 and phosphate buffer saline (PBS. Filtrates obtained were concentrated in a rotary evaporator and lyophilized. Antimicrobial activity of the extracts, at a concentration of 25 mg/ml was tested against four bacterial isolates using agar well diffusion method. Phytochemical analysis of the plant extract for the presence of tannins, saponins, alkaloids and flavanoids was based on chemical examination. Results and discussions: Extracts from methanol-water mixture showed some antimicrobial activity against Lactobacillus brevis (NCIMB 4617, L. plantarum (NCDO 1752 and Escherichia coli and Proteus vulgaris. The minimum inhibitory concentration (MIC of extracts ranged between 1.57 and 12.5 mg/ml except in E. coli with value greater than 12.5.mg/ml. The phytochemical screening indicated that the extract tested positive for tannin, saponin, flavonoids and alkaloids. Results showed that F. zanthoxyloides would be valuable in the treatment of microbial diseases particularly those of the oral cavity. The considerable antibacterial activities exhibited by the extract of the plant thus justify the use as a teeth cleansing agent in the local setting. The problem of antibiotics resistance to synthetic drugs is also expected to be mitigated with the use of natural drugs. [J Intercult Ethnopharmacol 2012; 1(1.000: 1-6

  12. Isolation and cultivation of microalgae select for low growth rate and tolerance to high pH

    DEFF Research Database (Denmark)

    Berge, Terje; Daugbjerg, Niels; Hansen, Per Juel

    2012-01-01

    Harmful microalgal blooms or red tides are often associated with high levels of pH. Similarly, species and strains of microalgae cultivated in the laboratory with enriched media experience recurrent events of high pH between dilutions with fresh medium. To study the potential for laboratory...... of upper pH tolerance limits were higher in the younger (20 years). These results suggest selection of strains best adapted to tolerate or postpone/avoid events of high pH in the laboratory. Our data have implications for experimental studies of pH response and reaction norms in general of microalgae...

  13. A coordination polymer based magnetic adsorbent material for hemoglobin isolation from human whole blood, highly selective and recoverable

    Science.gov (United States)

    Zhang, Xiaoxing; Tan, Jipeng; Xu, Xinxin; Shi, Fanian; Li, Guanglu; Yang, Yiqiao

    2017-09-01

    A composite material has been obtained successfully through the loading of nanoscale coordination polymer on magnetic Fe3O4@SiO2 core-shell particle. In this composite material, coordination polymer nanoparticles distribute uniformly on Fe3O4@SiO2 and these two components are ;tied; together firmly with chemical bonds. Adsorption experiments suggest this composite material exhibits very excellent selectivity to hemoglobin. But under the same condition, its adsorption to bovine serum albumin can almost be ignored. This selectivity can be attributed to the existence of hydrophobic interactions between coordination polymer nanoparticle and hemoglobin. For composite material, the hemoglobin adsorption process follows Langmuir model perfectly with high speed. The adsorbed hemoglobin can be eluted easily by sodium dodecyl sulfate stripping reagent with structure and biological activity of hemoglobin keeps well. The composite material was also employed to separate hemoglobin from human whole blood, which receives a very satisfactory result. Furthermore, magnetic measurement reveals ferromagnetic character of this composite material with magnetization saturation 3.56 emu g-1 and this guarantees its excellent magnetic separation performance from the treated solution.

  14. Repeated Aspergillus isolation in respiratory samples from non-immunocompromised patients not selected based on clinical diagnoses: colonisation or infection?

    Directory of Open Access Journals (Sweden)

    Barberan Jose

    2012-11-01

    Full Text Available Abstract Background Isolation of Aspergillus from lower respiratory samples is associated with colonisation in high percentage of cases, making it of unclear significance. This study explored factors associated with diagnosis (infection vs. colonisation, treatment (administration or not of antifungals and prognosis (mortality in non-transplant/non-neutropenic patients showing repeated isolation of Aspergillus from lower respiratory samples. Methods Records of adult patients (29 Spanish hospitals presenting ≥2 respiratory cultures yielding Aspergillus were retrospectively reviewed and categorised as proven (histopathological confirmation or probable aspergillosis (new respiratory signs/symptoms with suggestive chest imaging or colonisation (symptoms not attributable to Aspergillus without dyspnoea exacerbation, bronchospasm or new infiltrates. Logistic regression models (step–wise were performed using Aspergillosis (probable + proven, antifungal treatment and mortality as dependent variables. Significant (p 2 were considered. Results A total of 245 patients were identified, 139 (56.7% with Aspergillosis. Aspergillosis was associated (R2 = 0.291 with ICU admission (OR = 2.82, congestive heart failure (OR = 2.39 and steroids pre-admission (OR = 2.19 as well as with cavitations in X-ray/CT scan (OR = 10.68, radiological worsening (OR = 5.22 and COPD exacerbations/need for O2 interaction (OR = 3.52. Antifungals were administered to 79.1% patients with Aspergillosis (100% proven, 76.8% probable and 29.2% colonised, with 69.5% patients receiving voriconazole alone or in combination. In colonised patients, administration of antifungals was associated with ICU admission at hospitalisation (OR = 12.38. In Aspergillosis patients its administration was positively associated (R2 = 0.312 with bronchospasm (OR = 9.21 and days in ICU (OR = 1.82 and negatively with Gold III + IV (OR = 0.26, stroke

  15. Sensitive and selective culture medium for detection of environmental Clostridium difficile isolates without requirement for anaerobic culture conditions.

    Science.gov (United States)

    Cadnum, Jennifer L; Hurless, Kelly N; Deshpande, Abhishek; Nerandzic, Michelle M; Kundrapu, Sirisha; Donskey, Curtis J

    2014-09-01

    Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Isolation of lactic acid bacteria from kuruma shrimp (Marsupenaeus japonicus) intestine and assessment of immunomodulatory role of a selected strain as probiotic.

    Science.gov (United States)

    Maeda, M; Shibata, A; Biswas, G; Korenaga, H; Kono, T; Itami, T; Sakai, M

    2014-04-01

    Fifty-one lactic acid bacteria (LAB) strains were isolated and identified based on 16S ribosomal DNA sequence from the intestinal tracts of 142 kuruma shrimps (Marsupenaeus japonicus) collected from Kanmon Strait, Fukuoka and Tachibana Bay, Nagasaki, Japan. Cellular immunomodulatory function of 51 isolated LAB strains was assessed by measuring the level of interferon (IFN)-γ induction in mouse spleen cell culture. The strain Lactococcus lactis D1813 exhibited the highest amount of IFN-γ production and also bactericidal activity and was selected for testing its immunomodulatory role as a probiotic in kuruma shrimp. We also assessed the effect of dietary incorporation of this probiotic on resistance to Vibrio penaeicida infection in the kuruma shrimp. Our results demonstrate that probiotic L. lactis D1813-containing diet-fed (10⁵ cfu g⁻¹) shrimps displayed a significant up-regulation of lysozyme gene expressions in the intestine and hepatopancreas. However, insignificantly higher expression of anti-lipopolysaccharide factor, super oxide dismutase, prophenoloxidase, and toll-like receptor 1 was recorded in the intestine of shrimps fed the probiotic diet. Moreover, significantly increased (P probiotic-incorporated diet compared with the control diet-fed group (28.3 %). The present study indicates the immunomodulatory role of the LAB L. lactis D1813 on the kuruma shrimp immune system and supports its potential use as an effective probiotic in shrimp aquaculture.

  17. Selection, isolation and growth kinetic study of a bacterial consortium obtained from the Potengi mangrove in the presence of crude oil

    Energy Technology Data Exchange (ETDEWEB)

    Costa, C.C.; Vaz, M.R.F.; Santos, E.S.; Macedo, G.R. [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Dept. de Engenharia Quimica], E-mail: natcintia@gmail.com; Costa, J.G. da [Universidade Federal do Amazonas (UFAM), Coari, AM (Brazil). Inst. de Saude e Biotecnologia

    2011-10-15

    The selection, isolation and kinetic study of a bacterial consortium obtained from a sample of soil from the Potengi mangrove, located in the city of Natal, Rio Grande do Norte, Brazil, has been carried out using the enrichment culture technique to observe aspects such as the evaluation of main growth parameters. The kinetic study used a rotary incubator shaker at 150rpm, under 30 deg C. The bacterial consortium isolated from the estuary of the Potengi River showed a good acclimation in minimum mineral medium with 1% (v/v) of oil. The cell concentration reached 2.55 g/L at 16h of cultivation and surface tension dropped. The maximum productivity in cells obtained was of 0.3 g/L.h, the specific velocity of growth was of 0.075h{sup -1}, with a generation time (tg) of 9.24h. This study seeks to demonstrate that the consortium can be used as inoculants in biological treatments, capable of reducing the waste's degradation time. (author)

  18. Simultaneous Counter-Ion Co-Deposition a Technique Enabling Matrix Isolation Spectroscopy Studies Using Low-Energy Beams of Mass-Selected Ions

    Science.gov (United States)

    Ludwig, Ryan M.; Moore, David T.

    2014-06-01

    Matrix isolation spectroscopy was first developed in Pimentel's group during the 1950's to facilitate spectroscopic studies of transient species. Cryogenic matrices of condensed rare gases provide an inert chemical environment with facile energy dissipation and are transparent at all wavelengths longer than vacuum UV, making them ideal for studying labile and reactive species such as radicals, weakly bound complexes, and ions. Since frozen rare gases are poor electrolytes, studies of ions require near-equal populations of anions and cations in order to stabilize the number densities required for spectroscopic experiments. Many techniques for generation of ions for using in matrix isolation studies satisfy this criterion intrinsically, however when ion beams generated in external sources are deposited, the counter-ions typically arise via secondary processes that are at best loosely controlled. It has long been recognized that it would be desirable to stabilize deposition of mass-selected ions generated in an external source using simultaneous co-deposition of a beam of counter-ions, however previous attempts to achieve this have been reported as unsuccessful. The Moore group at Lehigh has demonstrated successful experiments of this type, using mass-selected anions generated from a metal cluster source, co-deposited with a balanced current of cations generated in a separate electron ionization source. This talk will focus on the details of the technique, and present some results from proof-of-concept studies on anionic copper carbonyl complexes formed in argon matrices following co-deposition of Cu- with Ar+ or Kr+. Funding support from NSF CAREER Award CHE-0955637 is gratefully acknowledged. Whittle et al., J. Chem. Phys. 22, p.1943 (1954); Becker et al., J. Chem. Phys. 25, p.224 (1956). Godbout et al., J. Chem. Phys. 96, p.2892 (1996). Sabo et al., Appl. Spectrosc. 45, p. 535 (1991).

  19. Selective block of IKs plays a significant role in MAP triangulation induced by IKr block in isolated rabbit heart.

    Science.gov (United States)

    Guérard, N C; Traebert, M; Suter, W; Dumotier, B M

    2008-01-01

    The role of IKr (rapidly-activating delayed rectifier K(+) current) block in triangulation of monophasic action potentials (MAP) and in development of torsade de pointes (TdP) arrhythmia is known. Combined IKr and IKs (slowly-activating delayed rectifier K(+) current) block has been demonstrated to promote TdP. The aim of this study was to describe a possible implication of IKs block in MAP triangulation. Four contact electrodes were placed on the epicardium of the left ventricle of Langendorff-perfused rabbit hearts to record monophasic action potentials (MAP), with an IKr blocker d,l-sotalol (3 to 100 microM, n=6) or a non-selective IKr blocker, quinidine (1 to 30 microM, n=6). Their effects were assessed with or without a specific IKs blocker chromanol 293B (20 microM, n=6), on MAP duration at 30, 60 and 90% of repolarization (APD30, 60 and 90, respectively) and MAP triangulation (APD90-APD30) at 1 and 0.2 Hz. D,L-sotalol increased significantly APD90 and triangulation with reverse use-dependency for concentrations > or =10 microM. Quinidine markedly prolonged APD90 and triangulation with reverse use-dependency at concentrations > or =3 microM. Chromanol 293B alone had no effects on APD, but when combined with D,L-sotalol or quinidine (i) increased APD prolonging effects, (ii) lowered values of pro-arrhythmic concentrations, (iii) increased incidence and length of D,L-sotalol- or quinidine-induced Early Afterdepolarizations (EADs) and TdP. All these events were primarily due to an important slowing of final repolarization, i.e. a marked increased triangulation. IKs, even of low amplitude in rabbits, plays a key role in ventricular repolarization. IKs is involved in prolonged MAP duration mainly by triangulation and subsequent increased drug arrhythmogenicity. Therefore drug affinity for IKs must be evaluated with IKr studies as part of preclinical drug cardiac safety assessment.

  20. Premating isolation is determined by larval rearing substrates in cactophilic Drosophila mojavensis. IX. Host plant and population specific epicuticular hydrocarbon expression influences mate choice and sexual selection.

    Science.gov (United States)

    Havens, J A; Etges, W J

    2013-03-01

    Sexual signals in cactophilic Drosophila mojavensis include cuticular hydrocarbons (CHCs), contact pheromones that mediate female discrimination of males during courtship. CHCs, along with male courtship songs, cause premating isolation between diverged populations, and are influenced by genotype × environment interactions caused by different host cacti. CHC profiles of mated and unmated adult flies from a Baja California and a mainland Mexico population of D. mojavensis reared on two host cacti were assayed to test the hypothesis that male CHCs mediate within-population female discrimination of males. In multiple choice courtship trials, mated and unmated males differed in CHC profiles, indicating that females prefer males with particular blends of CHCs. Mated and unmated females significantly differed in CHC profiles as well. Adults in the choice trials had CHC profiles that were significantly different from those in pair-mated adults from no-choice trials revealing an influence of sexual selection. Females preferred different male CHC blends in each population, but the influence of host cactus on CHC variation was significant only in the mainland population indicating population-specific plasticity in CHCs. Different groups of CHCs mediated female choice-based sexual selection in each population suggesting that geographical and ecological divergence has the potential to promote divergence in mate communication systems. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

  1. Effectively designed molecularly imprinted polymers for selective isolation of the antidiabetic drug metformin and its transformation product guanylurea from aqueous media

    Energy Technology Data Exchange (ETDEWEB)

    Kyzas, George Z.; Nanaki, Stavroula G. [Laboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Koltsakidou, Anastasia; Papageorgiou, Myrsini; Kechagia, Maria [Laboratory of Environmental Pollution Control, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Bikiaris, Dimitrios N. [Laboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece); Lambropoulou, Dimitra A., E-mail: dlambro@chem.auth.gr [Laboratory of Environmental Pollution Control, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki (Greece)

    2015-03-25

    Highlights: • Preparation of novel MIPs with remarkable recognition properties. • Selective isolation and removal of metformin and guanylurea from aqueous media. • Detailed adsorbent characterization and adsorption studies. • Successful application of synthesized MIPs as dispersive SPE sorbents. • Estimation of expanded uncertainty following a bottom-up approach. - Abstract: In the present study, two novel molecularly imprinted polymers (MIPs) with remarkable recognition properties for metformin and its transformation product, guanylurea, have been prepared for their selective, enrichment, isolation and removal from aqueous media. The prepared adsorbents were characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and swelling experiments. The performance of the prepared MIPs was evaluated by various parameters including the influence of pH, contact time, temperature and initial compound concentration. The effects on the adsorption behavior of the removal process parameters were studied and the equilibrium data were fitted by the Langmuir and Freundlich models. Due to the imprinting effect, adsorption performance of MIPs was always superior to its corresponding NIP (non-imprinted polymer), with maximum adsorption capacity ∼80 mg g{sup −1} for both MIPs. Stability and reusability of the MIPs up to the 5th cycle meant that they could be applied repeatedly without losing substantial removal ability. In the next step, the prepared MIP nanoparticles were evaluated as sorbents in a dispersive solid phase extraction (D-SPE) configuration for selective enrichment and determination of metformin and guanylurea in different aqueous matrices. Under the working extraction conditions, the D-SPE method showed good linearity in the range of 50–1000 ng L{sup −1}, repeatability of the extractions (RSD 2.1–5.1%, n = 3), and low limits of detection (1.5–3.4 ng L{sup −1}). The expanded uncertainty of the data obtained

  2. Analog signal isolation techniques

    Energy Technology Data Exchange (ETDEWEB)

    Beadle, E.R.

    1992-12-31

    This paper discusses several techniques for isolating analog signals in an accelerator environment. The techniques presented here encompass isolation amplifiers, voltage-to-frequency converters (VIFCs), transformers, optocouplers, discrete fiber optics, and commercial fiber optic links. Included within the presentation of each method are the design issues that must be considered when selecting the isolation method for a specific application.

  3. Analog signal isolation techniques

    Energy Technology Data Exchange (ETDEWEB)

    Beadle, E.R.

    1992-01-01

    This paper discusses several techniques for isolating analog signals in an accelerator environment. The techniques presented here encompass isolation amplifiers, voltage-to-frequency converters (VIFCs), transformers, optocouplers, discrete fiber optics, and commercial fiber optic links. Included within the presentation of each method are the design issues that must be considered when selecting the isolation method for a specific application.

  4. Genetic drift vs. natural selection in a long-term small isolated population: major histocompatibility complex class II variation in the Gulf of California endemic porpoise (Phocoena sinus).

    Science.gov (United States)

    Munguia-Vega, Adrian; Esquer-Garrigos, Yareli; Rojas-Bracho, Lorenzo; Vazquez-Juarez, Ricardo; Castro-Prieto, Aines; Flores-Ramirez, Sergio

    2007-10-01

    Although many studies confirm long-term small isolated populations (e.g. island endemics) commonly sustain low neutral genetic variation as a result of genetic drift, it is less clear how selection on adaptive or detrimental genes interplay with random forces. We investigated sequence variation at two major histocompatibility complex (Mhc) class II loci on a porpoise endemic to the upper Gulf of California, México (Phocoena sinus, or vaquita). Its unique declining population is estimated around 500 individuals. Single-strand conformation polymorphism analysis revealed one putative functional allele fixed at the locus DQB (n = 25). At the DRB locus, we found two presumed functional alleles (n = 29), differing by a single nonsynonymous nucleotide substitution that could increase the stability at the dimer interface of alphabeta-heterodimers on heterozygous individuals. Identical trans-specific DQB1 and DRB1 alleles were identified between P. sinus and its closest relative, the Burmeister's porpoise (Phocoena spinipinnis). Comparison with studies on four island endemic mammals suggests fixation of one allele, due to genetic drift, commonly occurs at the DQA or DQB loci (effectively neutral). Similarly, deleterious alleles of small effect are also effectively neutral and can become fixed; a high frequency of anatomical malformations on vaquita gave empirical support to this prediction. In contrast, retention of low but functional polymorphism at the DRB locus was consistent with higher selection intensity. These observations indicated natural selection could maintain (and likely also purge) some crucial alleles even in the face of strong and prolonged genetic drift and inbreeding, suggesting long-term small populations should display low inbreeding depression. Low levels of Mhc variation warn about a high susceptibility to novel pathogens and diseases in vaquita.

  5. Selection and identification of oleaginous yeast isolated from soil, animal feed and ruminal fluid for use as feed supplement in dairy cattle.

    Science.gov (United States)

    Paserakung, A; Pattarajinda, V; Vichitphan, K; Froetschel, M A

    2015-10-01

    The purpose of this study was to select oleaginous yeast for microbial lipid production. Sixty-four yeast isolates were obtained from soil (GSY1-12), animal feeds (FDY1-21), and ruminal fluid (RMY1-31) using yeast extract peptone dextrose (YPD) agar. The cultivation of these isolates on nitrogen limited-medium revealed that GSY2 to GSY6, GSY10, FDY2, FDY12 and FDY14 accumulated lipid over 20% of dry biomass. Therefore, they were preliminarily classified as oleaginous yeast. In subsequent experiment, an 8 × 3 factorial in completely randomized design was conducted to examine the effect of eight oleaginous yeast strains and three nitrogen sources (peptone, (NH4 )2 SO4 , urea) on lipid accumulation when using molasses as substrate. The result illustrated that only GSY3 and GSY10 accumulated lipid over 20% of biomass when using peptone or (NH4 )2 SO4 but urea did not. However, GSY10 gave higher biomass and lipid yield than GSY3 (P < 0·05). Identification of GSY10 using 26S rDNA illustrated that GSY10 belongs to Trichosporon asahii. Fatty acid profiles of this strain contained unsaturated fats up to 62·5% of which oleic acid (C18:1 ) was predominant. In conclusion, T. asahii GSY10 was the most promising oleaginous yeast for microbial lipid production from molasses. This study illustrated the ability of T. asahii GSY10 to utilize molasses and (NH4 )2 SO4 for synthesizing and accumulating cellular lipid of which oleic acid (C18:1 ) was predominant. This yeast would be used for microbial lipid production used as feed supplement in dairy cattle. © 2015 The Society for Applied Microbiology.

  6. Antibiotic resistance, ability to form biofilm and susceptibility to copper alloys of selected staphylococcal strains isolated from touch surfaces in Polish hospital wards.

    Science.gov (United States)

    Różańska, Anna; Chmielarczyk, Agnieszka; Romaniszyn, Dorota; Bulanda, Małgorzata; Walkowicz, Monika; Osuch, Piotr; Knych, Tadeusz

    2017-01-01

    Despite the employment of sanitary regimes, contact transmission of the aetiological agents of hospital infections is still exceedingly common. The issue of microbe transmission becomes particularly important when facing multidrug-resistant microorganisms such as methicillin-resistant staphylococci. In the case of deficiencies in cleaning and disinfection procedures, hospital equipment made of copper alloys can play an important role, complementing traditional hospital hygiene procedures. The objective of this study was to characterize staphylococcal strains isolated from touch surfaces in Polish hospital wards in terms of their drug resistance, ability to form biofilm and susceptibility to antimicrobial activity of copper alloys. The materials for the study were 95 staphylococcal strains isolated from touch surfaces in 13 different hospital wards from Małopolska province (the south of Poland). Phenotypic and genotypic antibiotic resistance were checked for erythromycin, clindamycin, gentamycin, ciprofloxacin, trimethoprim/sulfamethoxazole and mupirocin. Biofilm formation ability for the tested strains was checked with the use of culture on Congo red agar. Susceptibility to copper, tin bronze, brass and new silver was tested using a modification of the Japanese standard. Over 67% of the analysed staphylococcal strains were methicillin-resistant (MR). Four strains were resistant to all of the tested antibiotics, and 14 were resistant to all except mupirocin. Strains classified as MR had significantly increased resistance to the remaining antibiotic groups. About one-third of the analysed strains revealed biofilm-forming ability. Among the majority of species, biofilm-forming and non-biofilm-forming strains were distributed evenly; in the case of S. haemolyticus only, negative strains accounted for 92.8%. Susceptibility to copper alloys was different between strains and rather lower than in the case of the SA strain selected for comparison. Coagulase

  7. Selective Toxicity of Persian Gulf Sea Cucumber (Holothuria parva) and Sponge (Haliclona oculata) Methanolic Extracts on Liver Mitochondria Isolated from an Animal Model of Hepatocellular Carcinoma.

    Science.gov (United States)

    Seydi, Enayatollah; Motallebi, Abbasali; Dastbaz, Maryam; Dehghan, Sahar; Salimi, Ahmad; Nazemi, Melika; Pourahmad, Jalal

    2015-12-01

    Natural products isolated from marine environments are well known for their pharmacodynamic potential in diverse disease treatments, such as for cancer or inflammatory conditions. Sea cucumbers are marine animals of the phylum Echinoderm and the class Holothuroidea, with leathery skin and gelatinous bodies. Sponges are important components of Persian Gulf animal communities, and the marine sponges of the genus Haliclona have been known to display broad-spectrum biological activity. Many studies have shown that sea cucumbers and sponges contain antioxidants and anti-cancer compounds. This study was designed to determine the selective toxicity of Persian Gulf sea cucumber (Holothuria parva) and sponge (Haliclona oculata) methanolic extracts on liver mitochondria isolated from an animal model of hepatocellular carcinoma, as part of a national project that hopes to identify novel potential anticancer candidates among Iranian Persian Gulf flora and fauna. To induce hepatocarcinogenesis, rats were given diethylnitrosamine (DEN) injections (200 mg/kg i.p. by a single dose), and then the cancer was promoted with 2-acetylaminofluorene (2-AAF) (0.02 w/w) for two weeks. Histopathological evaluations were performed, and levels of liver injury markers and a specific liver cancer marker (alpha-fetoprotein), were determined for confirmation of hepatocellular carcinoma induction. Finally, mitochondria were isolated from cancerous and non-cancerous hepatocytes. Our results showed that H. parva methanolic extracts (250, 500, and 1000 µg/mL) and H. oculata methanolic extracts (200, 400, and 800 µg/mL) increased reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP), mitochondrial swelling, and cytochrome c release in the mitochondria obtained from cancerous hepatocytes, but not in mitochondria obtained from non-cancerous liver hepatocytes. These extracts also induced caspase-3 activation, which is known as a final mediator of apoptosis, in the hepatocytes

  8. Isolamento e seleção de rizobactérias promotoras do crescimento de Pinus taeda Isolation and selection of Pinus taeda growth promoting rhizobacteria

    Directory of Open Access Journals (Sweden)

    Juliana Margarido Fonseca Couto Brunetta

    2010-06-01

    Full Text Available Este trabalho teve como objetivos isolar e selecionar rizobactérias promotoras do crescimento de P. taeda. O isolamento foi realizado a partir de mudas com 150 dias de idade, realizando-se a seleção por meio da inoculação dos isolados (10(8 u.f.c mL-1 na proporção de 0,1 mL de inóculo.cm-3 de substrato. Aos 150 dias de semeadura, avaliaram-se as seguintes variáveis: altura da parte aérea, diâmetro do coleto e pesos de matéria seca de raízes e da parte aérea. Entre 99 isolados testados em P. taeda, apenas seis (UFV-AL9, UFV-AM5, UFV-AM2, UFV-F3, UFV-G2 e UFV-G4 destacaram-se quanto à indução de crescimento e ao melhor índice de qualidade de muda. Não se observaram diferenças significativas entre a testemunha e os demais isolados testados.The objectives of this research were to isolate and select P. taeda growth promoting rhizobacteria. The bacteria were isolated from 150 day-old pine seedlings and the selection was carried out with the strains inoculation (10(8 u.f.c/mL in the proportion of 0.1 mL/cm³. After 150 days of sowing, aerial part height, stem diameter and roots and aerial part dry matter weight were evaluated. From the 99 tested strains, six (UFV AL9, UFV-AM5, UFV-AM2, UFV-F3, UFV-G2 and UFV-G4 stood out due to their capacity of stimulating the growth and quality improvement of P. taeda seedlings. Significant differences were not observed between the control and the other tested strains.

  9. Holoprosencephaly: Characterization of the deletion of 21q22.3 and isolation of cDNAs by a direct selection technique

    Energy Technology Data Exchange (ETDEWEB)

    Yamakawa, K.; Colbern, S.; Brusilovsky, A. [Univ. of California, Los Angeles, CA (United States)] [and others

    1994-09-01

    Holoprosencephaly (HP) is characterized by impaired cleavage of the embryonic forebrain and incomplete mid-facial development. The etiology is heterogeneous and may include aneuploidies for chromosomes 2, 3, 7, 13, 18 and 21. We have narrowed the chromosome 21 candidate region by analyzing 2 cases of HP with deletion 21q22 using FISH and Southern blot techniques. For the smaller deletion, the regions for D21S25, D21S154, D21S171 and D21S44 were deleted and for D21S42 was not. Combining these data with previous reports of deletion of 21q22.3 (ColVIA2-ter) without the holoprosencephaly phenotype indicate that the region responsible for holoprosencephaly spans the 2-3 Mb region including PFKL and ITGB2 (CD18) that has also been linked to progressive myoclonus epilepsy (EPM1). In order to isolate genes responsible for these diseases, we constructed a cDNA library from a 14-week trisomy 21 fetal brain using Uni-Zap XR (Stratagene). More than 95% clones have inserts ranging from 1-4 kb (ave. 2 kb). In addition we applied a direct cDNA selection method to BACs (Bacterial Artificial Chromosomes) in the 21q22.3 region. Using cDNA synthesized from trisomy 21 fetal brain, we attached Sau3AI linkers, digested with Sau3AI, attached second linkers and hybridized to biotinylated BAC DNAs which cover the candidate region. cDNA/BAC DNA hybrid molecules were captured on streptavidin-coated magnetic beads, non-specific cDNA were washed out, and specifically hybridized cDNA were eluted and amplified by PCR. Twice-selected PCR products were subcloned and analyzed. Southern blot analyses revealed that 21 out of 30 (70%) of fragments yielded unique bands on the original BACs. Eight clones contained repetitive sequences. We are now isolating cDNAs expressed in the Down syndrome fetal brain using these cDNA fragments. These genes now provide candidates for EPM1 and holoprosencephaly.

  10. Inhibitory activities of selected Sudanese medicinal plants on Porphyromonas gingivalis and matrix metalloproteinase-9 and isolation of bioactive compounds from Combretum hartmannianum (Schweinf) bark.

    Science.gov (United States)

    Mohieldin, Ebtihal Abdalla M; Muddathir, Ali Mahmoud; Mitsunaga, Tohru

    2017-04-20

    Periodontal diseases are one of the major health problems and among the most important preventable global infectious diseases. Porphyromonas gingivalis is an anaerobic Gram-negative bacterium which has been strongly implicated in the etiology of periodontitis. Additionally, matrix metalloproteinases-9 (MMP-9) is an important factor contributing to periodontal tissue destruction by a variety of mechanisms. The purpose of this study was to evaluate the selected Sudanese medicinal plants against P. gingivalis bacteria and their inhibitory activities on MMP-9. Sixty two methanolic and 50% ethanolic extracts from 24 plants species were tested for antibacterial activity against P. gingivalis using microplate dilution assay method to determine the minimum inhibitory concentration (MIC). The inhibitory activity of seven methanol extracts selected from the 62 extracts against MMP-9 was determined by Colorimetric Drug Discovery Kit. In search of bioactive lead compounds, Combretum hartmannianum bark which was found to be within the most active plant extracts was subjected to various chromatographic (medium pressure liquid chromatography, column chromatography on a Sephadex LH-20, preparative high performance liquid chromatography) and spectroscopic methods (liquid chromatography-mass spectrometry, Nuclear Magnetic Resonance (NMR)) to isolate and characterize flavogalonic acid dilactone and terchebulin as bioactive compounds. About 80% of the crude extracts provided a MIC value ≤4 mg/ml against bacteria. The extracts which revealed the highest potency were: methanolic extracts of Terminalia laxiflora (wood; MIC = 0.25 mg/ml) followed by Acacia totrtilis (bark), Ambrosia maritima (aerial part), Argemone mexicana (seed), C. hartmannianum (bark), Terminalia brownii (wood) and 50% ethanolic extract of T. brownii (bark) with MIC values of 0.5 mg/ml. T. laxiflora (wood) and C. hartmannianum (bark) which belong to combretaceae family showed an inhibitory activity over 50% at

  11. Genetic diversity and natural selection in the rhoptry-associated protein 1 (RAP-1) of recent Plasmodium knowlesi clinical isolates from Malaysia.

    Science.gov (United States)

    Rawa, Mira Syahfriena Amir; Fong, Mun-Yik; Lau, Yee-Ling

    2016-02-05

    The Plasmodium rhoptry-associated protein 1 (RAP-1) plays a role in the formation of the parasitophorous vacuole following the parasite's invasion of red blood cells. Although there is some evidence that the protein is recognized by the host's immune system, study of Plasmodium falciparum RAP-1 (PfRAP-1) suggests that it is not under immune pressure. A previous study on five old (1953-1962) P. knowlesi strains suggested that RAP-1 has limited genetic polymorphism and might be under negative selection. In the present study, 30 recent P. knowlesi isolates were studied to obtain a better insight into the polymorphism and natural selection of PkRAP-1. Blood samples from 30 knowlesi malaria patients were used. These samples were collected between 2010 and 2014. The PkRAP-1 gene, which contains two exons, was amplified by PCR, cloned into Escherichia coli and sequenced. Genetic diversity and phylogenetic analyses were performed using MEGA6 and DnaSP ver. 5.10.00 programs. Thirty PkRAP-1 sequences were obtained. The nucleotide diversity (π) of exons 1, 2 and the total coding region (0.00915, 0.01353 and 0.01298, respectively) were higher than those of the old strains. Further analysis revealed a lower rate of non-synonymous (dN) than synonymous (dS) mutations, suggesting negative (purifying) selection of PkRAP-1. Tajima's D test and Fu and Li's D test values were not significant. At the amino acid level, 22 haplotypes were established with haplotype H7 having the highest frequency (7/34, 20.5 %). In the phylogenetic analysis, two distinct haplotype groups were observed. The first group contained the majority of the haplotypes, whereas the second had fewer haplotypes. The present study found higher genetic polymorphism in the PkRAP-1 gene than the polymorphism level reported in a previous study. This observation may stem from the difference in sample size between the present (n = 30) and the previous (n = 5) study. Synonymous and non-synonymous mutation analysis indicated

  12. Remineralization effects of casein phosphopeptide-amorphous calcium phosphate crème on artificial early enamel lesions of primary teeth.

    Science.gov (United States)

    Zhang, Qiong; Zou, Jing; Yang, Ran; Zhou, Xuedong

    2011-09-01

    Caries in children younger than 72 months is called early childhood caries (ECC). Sixty-six per cent of Chinese children younger than 5 years old have dental decay, and about 97% of them are untreated. This in vitro study was conducted to evaluate the remineralization effects of the casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) crème on the artificial early enamel lesions of the primary teeth and to assess its caries-prevention efficiency. Enamel specimens with artificial early lesions were produced and were then randomly divided into Group A: distilled and deionized water, DDW, as negative control; Group B: CPP-ACP crème, test group; Group C: 500 ppm NaF solution, as positive control. The enamel surface microhardness (SMH) was measured before, after demineralization, and 30 days after remineralization. The results were analysed with the SPSS 13.0 software package. The enamel specimens were analysed by the scanning electron microscope. The CPP-ACP crème increased SMH of the eroded enamel significantly more than 500 ppm NaF solution did. The morphology of the enamel was different in each group. The CPP-ACP crème is effective in remineralizing early enamel lesions of the primary teeth, a little more effective than 500 ppm NaF and can be used for the prevention of ECC. 2011 The Authors. International Journal of Paediatric Dentistry © 2011 BSPD, IAPD and Blackwell Publishing Ltd.

  13. An Innovative Approach to Treat Incisors Hypomineralization (MIH: A Combined Use of Casein Phosphopeptide-Amorphous Calcium Phosphate and Hydrogen Peroxide—A Case Report

    Directory of Open Access Journals (Sweden)

    Stefano Mastroberardino

    2012-01-01

    Full Text Available Molar Incisor Hypomineralization (MIH is characterized by a developmentally derived deficiency in mineral enamel. Affected teeth present demarcated enamel opacities, ranging from white to brown; also hypoplasia can be associated. Patient frequently claims aesthetic discomfort if anterior teeth are involved. This problem leads patients to request a bleaching treatment to improve aestheticconditions.Nevertheless, hydrogen peroxide can produce serious side-effects, resulting from further mineral loss. Microabrasion and/or a composite restoration are the treatments of choice in teeth with mild/moderate MIH, but they also need enamel loss. Recently, a new remineralizing agent based on Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP has been proposed to be effective in hypomineralized enamel, improving also aesthetic conditions. The present paper presents a case report of a young man with white opacities on incisors treated with a combined use of CPP-ACP mousse and hydrogen peroxide gel to correct the aesthetic defect. The patient was instructed to use CPP-ACP for two hours per day for three months in order to obtain enamel remineralization followed by a combined use of CPP-ACP and bleaching agent for further two months. At the end of this five-month treatment, a noticeable aesthetic improvement of the opacities was observed.

  14. Effects of the addition of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on mechanical properties of luting and lining glass ionomer cement

    Science.gov (United States)

    Heravi, Farzin; Bagheri, Hossein; Rangrazi, Abdolrasoul; Mojtaba Zebarjad, Seyed

    2016-07-01

    Recently, the addition of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into glass ionomer cements (GICs) has attracted interest due to its remineralization of teeth and its antibacterial effects. However, it should be investigated to ensure that the incorporation of CPP-ACP does not have significant adverse effects on its mechanical properties. The purpose of this study was to evaluate the effects of the addition of CPP-ACP on the mechanical properties of luting and lining GIC. The first step was to synthesize the CPP-ACP. Then the CPP-ACP at concentrations of 1%, 1.56% and 2% of CPP-ACP was added into a luting and lining GIC. GIC without CPP-ACP was used as a control group. The results revealed that the incorporation of CPP-ACP up to 1.56%(w/w) increased the flexural strength (29%), diametral tensile strength (36%) and microhardness (18%), followed by a reduction in these mechanical properties at 2%(w/w) CPP-ACP. The wear rate was significantly decreased (23%) in 1.56%(w/w) concentration of CPP-ACP and it was increased in 2%(w/w). Accordingly, the addition of 1.56%(w/w) CPP-ACP into luting and lining GIC had no adverse effect on the mechanical properties of luting and lining GIC and could be used in clinical practice.

  15. Evaluation of the mechanical properties of conventional glass ionomer cement after the addition of casein phosphopeptide amorphous calcium phosphate: An in vitro study

    Directory of Open Access Journals (Sweden)

    Shalini Aggarwal

    2014-01-01

    Full Text Available Background: Casein phosphopeptides-amorphous calcium phosphate (CPP-ACP products have gained much importance in restorative dentistry and minimally invasive dentistry. Addition of CPP into glass ionomer cement (GIC has been shown to interact with fluoride ions to produce an additive anticariogenic effect through the formation of stabilized amorphous calcium fluoride phosphate phase. Aim: The aim of this study was to determine the additive effect of CPP-ACP on the mechanical properties of conventional GIC. Materials and Methods: The control GIC was prepared with self-curing GIC. The GIC containing CPP-ACP was prepared from the same batch, with 1.56% w/w CPP-ACP incorporated. Compressive strength and microtensile bond strength tests were done. Energy dispersive X-ray (EDX analysis was used to determine the composition of various structural phases. Results: Incorporation of 1.56% w/w CPP-ACP into the GIC resulted in an increase in compressive strength and microtensile bond strength. The representative EDX spectra taken showed enhanced release of calcium, phosphate, and fluoride ions.

  16. Effect of casein phosphopeptide-amorphous calcium phosphate (cpp-acp on caries-like lesions in terms of time and nano-hardness: An in vitro study

    Directory of Open Access Journals (Sweden)

    Shanthan Mettu

    2015-01-01

    Full Text Available Context: Time bound increase in the nanohardness of the enamel after remineralization with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP in a regular interval of 1 h has not been explored in the literature to a greater extent. Aims: To determine and compare the maximum hardness of the remineralized caries-like lesions, in terms of nanohardness and the rate of achieving maximum hardness at 1-h interval, after treatment with artificial saliva and CPP-ACP, over 12 h. Materials and Methods: Fifty longitudinal sections of extracted sound permanent maxillary central, lateral incisors were immersed in demineralizing solution for 4 days. The samples were then randomly divided into three groups, consisting of 12 sections each for soaking them in three different media-isotonic saline, artificial saliva, and CPP-ACP for 12 h. The nanohardness was measured on the labial surface, at baseline, after erosion, and after remineralization at 1-h interval. Statistical Analysis Used: The data was analyzed with paired t-test, one-way analysis of variance (ANOVA and post-hoc analysis. Results: CPP-ACP increased the enamel hardness significantly (P < 0.001, at an increased rate, than artificial saliva. Conclusions: This study has provided an insight into the frequency of use of CPP-ACP, once per day, as the nanohardness of enamel samples increased within 1 h of application and remained within the normal limits after 12 h.

  17. Efficacy of a casein phosphopeptide amorphous calcium phosphate (CPP-ACP) paste in preventing white spot lesions in patients with fixed orthodontic appliances: A prospective clinical trial.

    Science.gov (United States)

    Esenlik, E; Uzer Çelik, E; Bolat, E

    2016-12-01

    The aim of this prospective study was to test the efficacy of casein phosphopeptide amorphous calcium phosphate (CPP-ACP) paste applied in-office to prevent white spot lesions (WSL) in patients undergoing fixed orthodontic treatment. Study design: this study was designed as a randomised controlled clinical trial. Fifty-seven patients undergoing nonextraction fixed orthodontic treatment were enrolled and divided randomly into two groups (control n = 28, experimental n = 29). The pretreatment plaque, gingival and bleeding indices, and oral hygiene habits were recorded. One group received CPP-ACP paste (GC Tooth Mousse) at each monthly orthodontic follow-up examination; the control group received routine orthodontic treatment. The mean patient age was 16.9 years in the experimental group and 17.1 years in the control group. The periodontal indices, decayed, missing, and filled teeth (DMFT), decayed, missing, and filled surfaces (DMFS), and the WSL prevalence pre- and post-treatment were measured and compared between the groups. differences between groups in normally distributed data between groups were assessed by the paired-t test, and the Wilcoxon Signed Rank test was used to compare variables that were not normally distributed. There was a lower incidence of WSL in the experimental group compared to the control group. Most of the WSL occurred at the maxillary incisors, mandibular canines and premolars. The in-office application of CPP-ACP paste did not prevent WSL development completely; however, it did significantly decrease the number of WSL compared to the control patients.

  18. The effect of casein phosphopeptide-amorphous calcium phosphate paste and sodium fluoride mouthwash on the prevention of dentine erosion: An in vitro study

    Science.gov (United States)

    Moezizadeh, Maryam; Alimi, Azar

    2014-01-01

    Aim: The purpose was to compare the effect of 0.2% sodium fluoride mouthwash and casein phosphopeptide-amorphous calcium phosphate paste on prevention of dentin erosion. Materials and Methods: Buccal surfaces of 36 sound premolar teeth were ground flat and polished with abrasive discs. Half the polished surfaces were covered with tape to maintain a reference surface. Samples were randomly allocated into three groups. Group A was pretreated with tooth mousse (TM) 4 times a day for 5 days. Group B was pretreated with 0.2% sodium fluoride mouthwash 4 times a day for 5 days. Group C was considered as the control group with no pretreatment. In the next step, the samples were exposed to Coca-Cola 4 times a day for 3 days. After each erosive cycle, the samples were rinsed with deionized water and stored in artificial saliva. The surface loss was determined using profilometry. Results: The erosion in both Groups A and B was less than the control group. The surface loss in mouthwash group was significantly lower than in the control group. Erosion in TM group was more than the mouthwash group and less than the control group. Conclusion: Sodium fluoride mouthwash is more effective for prevention of dentin erosion. PMID:24944448

  19. An Innovative Approach to Treat Incisors Hypomineralization (MIH): A Combined Use of Casein Phosphopeptide-Amorphous Calcium Phosphate and Hydrogen Peroxide—A Case Report

    Science.gov (United States)

    Mastroberardino, Stefano; Campus, Guglielmo; Strohmenger, Laura; Villa, Alessandro; Cagetti, Maria Grazia

    2012-01-01

    Molar Incisor Hypomineralization (MIH) is characterized by a developmentally derived deficiency in mineral enamel. Affected teeth present demarcated enamel opacities, ranging from white to brown; also hypoplasia can be associated. Patient frequently claims aesthetic discomfort if anterior teeth are involved. This problem leads patients to request a bleaching treatment to improve aestheticconditions.Nevertheless, hydrogen peroxide can produce serious side-effects, resulting from further mineral loss. Microabrasion and/or a composite restoration are the treatments of choice in teeth with mild/moderate MIH, but they also need enamel loss. Recently, a new remineralizing agent based on Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP) has been proposed to be effective in hypomineralized enamel, improving also aesthetic conditions. The present paper presents a case report of a young man with white opacities on incisors treated with a combined use of CPP-ACP mousse and hydrogen peroxide gel to correct the aesthetic defect. The patient was instructed to use CPP-ACP for two hours per day for three months in order to obtain enamel remineralization followed by a combined use of CPP-ACP and bleaching agent for further two months. At the end of this five-month treatment, a noticeable aesthetic improvement of the opacities was observed. PMID:23243519

  20. Do enamel microabrasion and casein phosphopeptide-amorphous calcium phosphate affect shear bond strength of orthodontic brackets bonded to a demineralized enamel surface?

    Science.gov (United States)

    Baysal, Asli; Uysal, Tancan

    2012-01-01

    To evaluate and compare the effects of enamel microabrasion, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and their combination on the shear bond strength (SBS) of orthodontic brackets bonded to demineralized enamel surface. One hundred human first premolar teeth were randomly allocated to one of five groups. Group I was considered as the control of other groups. For the remaining groups, demineralization was performed via solutions. In group II, brackets were directly bonded to the demineralized enamel surface. CPP-ACP was applied in group III, microabrasion was performed in group IV, and both microabrasion and CPP-ACP application were performed in group V. The specimens were tested for SBS. Bond failures were scored according to the Adhesive Remnant Index (ARI). Analysis of variance and Tukey tests were used to compare the SBS of the groups. ARI scores of the groups were evaluated with a G-test. The statistical significance was set at P Microabrasion and combination with CPP-ACP showed higher SBS compared to the control group. The differences between ARI scores of the groups were statistically significant (P microabrasion of the enamel, and the combination of these two methods improve the bonding to demineralized enamel.

  1. Novel sponge-like molecularly imprinted mesoporous silica material for selective isolation of bisphenol A and its analogues from sediment extracts

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jiajia [Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yun; Wang, Jincheng [Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China); Sun, Xiaoli [Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Shah, Syed Mazhar [Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China); Cao, Rong [Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Chen, Jiping, E-mail: chenjp@dicp.ac.cn [Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023 (China)

    2015-01-01

    Highlights: • Novel sponge-like molecularly imprinted mesoporous silica was synthesized. • Extraordinarily large specific surface area and highly interconnected 3-D porous network. • High specific adsorption capacity and fast adsorption kinetics for BPA. • Good class-selectivity and clean-up effect for bisphenols in sediment under SPE mode. • Good recoveries and sensitivity for bisphenols using the MISMS–SPE coupled with HPLC–DAD method. - Abstract: Bisphenol A (BPA) imprinted sponge mesoporous silica was synthesized using a combination of semi-covalent molecular imprinting and simple self-assembly process. The molecularly imprinted sponge mesoporous silica (MISMS) material obtained was characterized by FT-IR, scanning electron microscopy, transmission electron microscopy, and nitrogen adsorption–desorption measurements. The results show that the MISMS possessed a large specific surface area (850.55 m{sup 2} g{sup −1}) and a highly interconnected 3-D porous network. As a result, the MISMS demonstrated a superior specific adsorption capacity of 169.22 μmol g{sup −1} and fast adsorption kinetics (reaching equilibrium within 3 min) for BPA. Good class selectivity for BPA and its analogues (bisphenol F, bisphenol B, bisphenol E and bisphenol AF) was also demonstrated by the sorption experiment. The MISMS as solid-phase extraction (SPE) material was then evaluated for isolation and clean-up of these bisphenols (BPs) from sediment samples. An accurate and sensitive analytical method based on the MISMS–SPE coupled with HPLC–DAD has been successfully established for simultaneous determination of five BPs in river sediments with detection limits of 0.43–0.71 ng g{sup −1} dry weight (dw). The recoveries of BPs for lyophilizated sediment samples at two spiking levels (50 and 500 ng g{sup −1} dw for each BP) were in the range of 75.5–105.5% with RSD values below 7.5%.

  2. [Association of bioassays and molecular characterization to select new Bacillus thuringiensis isolates effective against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)].

    Science.gov (United States)

    Fatoretto, Júlio C; Sena, Janete A D; Barreto, Marliton R; Lemos, Manoel V F; Boiça, Arlindo L

    2007-01-01

    The fall armyworm, Spodoptera frugiperda (J. E. Smith), is one of the main corn pests and Bacillus thuringiensis is important in its control because of its entomopathogenic property. The objective of this study was the molecular characterization of B. thuringiensis isolates for cry1 locus presence and the assessment of the efficiency of these isolates in controlling S. frugiperda caterpillars. Gral-cry1 was used in the PCR analyses to confirm the presence of the cry1 locus in 15 isolates. A 3 x 10(8) spore/ml suspension bathed the diet used to feed 30 caterpillars per isolate, with three replications. The cry1 locus type genes of the different isolates were identified for five gene subclasses; linear regression analyses were carried out to ascertain possible associations between the presence of an individual cry1 locus gene and high levels of toxicity. All the DNAs amplified with Gral-cry1 presented an amplification product with the expected size. Regarding the levels of insecticide efficiency against the cob worm, 41 isolates presented 100% mortality and 16 presented an index between 70% and 90%. The cry1Ab gene was present in 80 isolates, cryb in 69 isolates, cry1Ac in all the isolates and cryv and cry1E in 93 and 27 isolates, respectively. The values regarding the individual effect of each gene on caterpillar mortality were significant at 1% probability for the cry1Ac and cry1E genes.

  3. Comparison of selective agars recommended by method ISO 11290-1 and chromogenic agars for the isolation of Listeria sp. in refrigerated sausages

    Directory of Open Access Journals (Sweden)

    Thalyta Marina Benetti

    2012-12-01

    Full Text Available The aim of this study was to determine the prevalence of Listeria sp. in refrigerated sausages, and to compare the performance of the selective plating media employed in the ISO 11290-1 method (PALCAM and Oxford agars with chromogenic agars (Chromogenic Listeria agars CM 1080 (OCLA and CM 1084. The prevalence of Listeria sp. detected was 52.9%, comprising 13.7% L. monocytogenes strains. The efficacy of the four agars for the isolation of L. monocytogenes proved to be satisfactory. Despite differences in composition of the chromogenic media assessed, these disparities did not affect concordance among results. However, PALCAM agar was shown to suppress other microorganisms more effectively, being more applicable for detecting Listeria strains present in lower quantities. Based on these results, the use of PALCAM agar, in combination with a chromogenic media, is recommended for enhanced isolation of atypical Listeria sp. strains in meat products.Este estudo teve como objetivo a análise da prevalência de Listeria sp. em linguiças resfriadas e a comparação dos meios seletivos utilizados no plaqueamento do método ISO 11290-1 (Ágar PALCAM e Ágar Oxford, e ágares cromogênicos (Ágares Listeria Cromogênico CM 1080 (OCLA e CM 1084 (ISO. A frequência de Listeria sp. foi de 52,9%, sendo que destas, 13,7% corresponderam à L. monocytogenes. A eficácia dos quatro ágares para o isolamento de L. monocytogenes demonstrou-se satisfatória. Apesar de haver algumas diferenças nas composições dos meios cromogênicos analisados, estas não pareceram influenciar nas concordâncias entre os resultados expressos. Contudo, o ágar PALCAM mostrou-se mais eficaz na supressão de outros micro-organismos, aumentando, assim, a possibilidade de detecção de espécies de Listeria presentes em número reduzido. Através deste trabalho sugere-se a utilização do ágar PALCAM associado a um meio cromogênico para aumentar a chance de isolamento de cepas at

  4. The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts.

    Science.gov (United States)

    Hill, P A; Murphy, G; Docherty, A J; Hembry, R M; Millican, T A; Reynolds, J J; Meikle, M C

    1994-11-01

    We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki < 20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants co-cultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10(-8) M) selective for the inhibition of human gelatinases A and B. CT435 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 alpha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are

  5. Characterization of a Group B Streptococcus infection based on the demographics, serotypes, antimicrobial susceptibility and genotypes of selected isolates from sterile and non-sterile isolation sites in three major hospitals in Malaysia.

    Science.gov (United States)

    Suhaimi, Mohd E S; Desa, Mohd N M; Eskandarian, Narges; Pillay, Stella G; Ismail, Zalina; Neela, Vasantha K; Masri, Siti N; Nordin, Syafinaz A

    The purpose of this study is to characterize GBS isolates that were collected from three major hospitals in a densely populated area of Klang Valley for their demographics, serotypes, antibiotic susceptibility patterns and genetic background. Sixty GBS isolates from sterile and non-sterile samples in three major hospitals in the Klang Valley area of Malaysia were collected by convenience sampling from 2012 until March 2014. These isolates were studied for their antimicrobial susceptibilities, serotypes and genotypes. Patients' demographic data and clinical information were collected from lab request forms. Diabetes mellitus was the only underlying condition (7 patients, 23.3%); the remaining samples were from patients who were immunocompromised due to medications. Fifty-nine (98%) isolates were sensitive to penicillin, while 78.3% and 88.3% of the isolates were sensitive to erythromycin and clindamycin, respectively. Serotype Ia was the most common serotype (n=27, 45%), followed by serotype III (n=10, 16.7%), V (n=9, 15%), VI (n=8, 13.3%), VIII (n=2, 3.3%) and VII (n=1, 1.7%). Random Amplified Polymorphic DNA (RAPD) typing showed a diverse genetic pedigree for all isolates, including four major groups that clustered according to geographical location. This preliminary study determines the prevalence of limited common serotypes and antimicrobial resistance in distinct GBS isolates. Nonetheless, the RAPD clustering pattern suggests a close genetic lineage of the GBS isolates based on their isolation sites and location of hospitals. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

  6. Prevalence of β-Lactamase Production among Pathogenic Bacteria Isolated from Surgical Site and Wound Infection amoung Patients Admitted in some selected Hospitals in Sokoto Metropolis, Nigeria

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    UK Muhammad

    2014-09-01

    Full Text Available Antimicrobial resistance among pathogenic bacteria is increasing worldwide especially against ß-lactam drugs, due to the production of ß-lactamase enzymes which destroy the ß-lactam ring of these antibiotics, thus preventing the action of penicillin binding proteins (PBPs. The prevalence of β-lactamase producing bacteria among patients admitted in three different hospitals were carried out in this study. The results of this study shows that out of one hundred and fifty one isolates obtained in three different hospitals in Sokoto metropolis, only 82 (54.0% were resistant to the antibiotics tested. These include 42 (51.2% were isolated in Usmanu Danfodiyo Teaching Hospital (UDUT, 26 (31.7% were isolated from Specialist Hospital Sokoto (S.H.S and 14 (17.1% were isolated from Maryam Abatcha Women and Children Hospital (MAWCH which has the least number of occurrence of the resistant isolates. β-lactamase test was carried out on the resistant isolates show s that out of the 82 isolates found resistant to the antibiotics tested, about 60 (73.2% were β- lactamase positive and the remaining 22 (26.8% were β-lactamase negative. Staphylococcus aureus has the highest resistant bacteria producing β-lactamase enzyme with 22 isolates, followed by Proteus mirabilis with 10 isolates. DOI: http://dx.doi.org/10.3126/ije.v3i3.11067 International Journal of Environment Vol.3(3 2014: 89-112

  7. Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate.

    Science.gov (United States)

    Yuangsaard, Napatchanok; Yongmanitchai, Wichien; Yamada, Mumoru; Limtong, Savitree

    2013-03-01

    Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH(4))(2)SO(4), 0.09 % yeast extract, 0.05 % KH(2)PO(4), and 0.05 % MgSO(4)·7H(2)O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.

  8. Casein phosphopeptides and CaCl2 increase penicillin production and cause an increment in microbody/peroxisome proteins in Penicillium chrysogenum.

    Science.gov (United States)

    Domínguez-Santos, Rebeca; Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Ibáñez, Ana; Morales, Alejandro; Martín, Juan-Francisco

    2017-03-06

    Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens). Calcium (Ca2+) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca2+ internalization. In this study we observe that the effect of CaCl2 and CPP is additive and promotes an increase in penicillin production of up to 10-12 fold. Combination of CaCl2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine β-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin. Penicillin biosynthesis in Penicillium chrysogenum is one of the best characterized secondary metabolism processes. However, the mechanism by which penicillin is secreted still remains to be elucidated. Taking into account the role played by Ca2+ and CPP in the secretory pathway and considering the positive effect that Ca2+ exerts on penicillin production, the analysis of global protein changes produced after CPP/CaCl2 addition is very helpful to decipher the processes related to the biosynthesis and

  9. Effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP and fluoride on the microhardness of enamel treated with a bleaching agent: ex vivo

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    Mügem Aslı Gürel Ekici

    2017-05-01

    Full Text Available Objective: The aim of this study was to compare the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP and 1.23% acidulated phosphate fluoride (APF; pH 3.5 on the microhardness of enamel treated with a bleaching agent. Materials and Method: Enamel slices (n=32; 2×4 mm were obtained from 8 mandibular permanent molar teeth. Specimens were embedded into acrylic resin blocks with the enamel surfaces facing upwards. Vickers microhardness (VHN values of the specimens were recorded at baseline. The specimens were randomly divided into 4 experimental groups, and the experimental designation was as follows: Group 1: no treatment (control, Group 2: 35% hydrogen peroxide (HP, Group 3: HP + CPP-ACP, Group 4: HP + APF application. After treatments, VHN values were measured and recorded again. Specimens were stored in artificial saliva at 37 °C for 1 week. After 1 week second application was done and VHN of the specimens was registered once more. Data were statistically analyzed with ANOVA and Tukey post hoc tests. Values obtained at baseline, and first and second applications were compared using paired samples t-test (α=0.05. Results: In inter-group comparisons, no statistically significant difference in the enamel microhardness values was found between the baseline, and first and second applications (p>0.05. In intra-group comparisons, again, no statistically significant difference in the enamel microhardness values was found between the baseline, and first and second applications (p>0.05. Conclusion: According to the limitations of this study it can be concluded that neither the HP application nor the CPP-ACP or APF application after HP had any significant effect on the enamel microhardness.

  10. Pre-Use Susceptibility to Ceftaroline in Clinical Staphylococcus aureus Isolates from Germany: Is There a Non-Susceptible Pool to be Selected?

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    Birgit Strommenger

    Full Text Available Ceftaroline is a new cephalosporin active against Methicillin-resistant Staphylococcus aureus (MRSA. Based on a representative collection of clinical S. aureus isolates from Germany, supplemented with isolates of clonal lineages ST228 and ST239, we demonstrate the in-vitro susceptibility towards ceftaroline prior to its introduction into clinical use for a total of 219 isolates. Susceptibility testing was performed by broth microdilution, disc diffusion and Etest, respectively. Results were interpreted according to EUCAST guidelines and showed considerable variance in dependence on clonal affiliation of the isolates tested. Among isolates of widespread hospital-associated lineages we found a high proportion of clinical isolates with MICs close to the EUCAST breakpoint (MIC50/90 1.0/1.5 mg/L; currently, interpretation of these "borderline" MICs is complicated by a lack of concordant susceptibility testing methods and reasonable breakpoint determination. Isolates of clonal lineages ST228 and ST239 demonstrated increased MIC50/90 values of 2.5/3.33 mg/L. Sequencing of mecA revealed no association of resistance to a specific mecA polymorphism, but rather reveals two regions in the non-penicillin-binding domain of PbP2a which displayed different combinations of mutations putatively involved in resistance development. This study provides national baseline data to (i adjust susceptibility testing methods and current breakpoints to clinical and epidemiological requirements, (ii evaluate current breakpoints with respect to therapeutic outcome and (iii monitor further resistance evolution.

  11. Selective in vitro and in silico butyrylcholinesterase inhibitory activity of diterpenes and rosmarinic acid isolated from Perovskia atriplicifolia Benth. and Salvia glutinosa L.

    Science.gov (United States)

    Senol, F Sezer; Ślusarczyk, Sylwester; Matkowski, Adam; Pérez-Garrido, Alfonso; Girón-Rodríguez, Francisco; Cerón-Carrasco, José P; den-Haan, Helena; Peña-García, Jorge; Pérez-Sánchez, Horacio; Domaradzki, Krzysztof; Orhan, Ilkay Erdogan

    2017-01-01

    Cholinesterase inhibition is one of the most treatment strategies against Alzheimer's disease (AD) where metal accumulation is also strongly associated with pathology of the disease. In the current study, we assessed inhibitory effect against acetyl- (AChE) and butyrylcholinesterase (BChE) and metal-chelating capacity of twelve diterpenes: arucadiol, miltirone, tanshinone IIa, 1-oxomiltirone, cryptotanshinone, 1,2-didehydromiltirone, 1,2-didehydrotanshinone IIa, 1β-hydroxycryptotanshinone, 15,16-dihydrotanshinone, tanshinone I, isotanshinone II, 1(S)-hydroxytanshinone IIa, and rosmarinic acid, isolated from Perovskia atriplicifolia and Salvia glutinosa. The compounds were tested at 10 μg/mL using ELISA microtiter assays against AChE and BChE. QSAR and molecular docking studies have been also performed on the active compounds. All of the compounds showed higher [e.g., IC50 = 1.12 ± 0.07 μg/mL for 1,2-didehydromiltirone, IC50 = 1.15 ± 0.07 μg/mL for cryptotanshinone, IC50 = 1.20 ± 0.03 μg/mL for arucadiol, etc.)] or closer [1,2-didehydrotanshinone IIa (IC50 = 5.98 ± 0.49 μg/mL) and 1(S)-hydroxytanshinone IIa (IC50 = 5.71 ± 0.27 μg/mL)] inhibition against BChE as compared to that of galanthamine (IC50 = 12.56 ± 0.37 μg/mL), whereas only 15,16-dihydrotanshinone moderately inhibited AChE (65.17 ± 1.39%). 1,2-Didehydrotanshinone IIa (48.94 ± 0.26%) and 1(S)-hydroxytanshinone IIa (47.18 ± 5.10%) possessed the highest metal-chelation capacity. The present study affords an evidence for the fact that selective BChE inhibitors should be further investigated as promising candidate molecules for AD therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Evaluation of antimicrobial activities of crude ethyl acetate and ethanol extracts of Morinda lucida leaf, stem, and bark growing in Adekunle Ajasin University Botanical Garden against selected clinical isolates

    OpenAIRE

    Osuntokun, Oludare T.

    2015-01-01

    The purpose of this research work is to determine the antimicrobial activities of crude ethyl acetate and ethanolic extracts of Morinda lucida leaf, stem, and bark growing in Adekunle Ajasin University Botanical Garden against selected clinical isolates. The plant Morindal lucida falls under the family Rubiaceae known to have wide usage in traditional medicine. Morinda lucida is a tropical West African tree of medium-size about 18–25 m tall, the bark is grey to brown in color, flowers are whi...

  13. Negative electron transfer dissociation of deprotonated phosphopeptide anions: choice of radical cation reagent and competition between electron and proton transfer.

    Science.gov (United States)

    Huzarska, Malwina; Ugalde, Israel; Kaplan, Desmond A; Hartmer, Ralf; Easterling, Michael L; Polfer, Nick C

    2010-04-01

    Despite significant developments in mass spectrometry technology in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a molecular open-shell cation is presented here as a possible reaction partner to induce electron transfer dissociation with deprotonated peptide anions. In this negative electron transfer dissociation (NETD) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C(16)H(10)(+*), which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the C(alpha)-C bond is observed to yield a and x fragments, similarly to electron detachment dissociation (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissociation process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe(+*) as the reagent cation results in sequential neutral losses (CO(2) and H(3)PO(4)) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissociation behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estimated at 2.5-4.5 eV, compared to 6.7-8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for anions.

  14. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies

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    Hirotsugu eFujitani

    2015-10-01

    Full Text Available Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representative of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member’s ecophysiology in a variety of habitats.

  15. Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method

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    J. Robert Smith

    2016-01-01

    Full Text Available Umbilical cord derived mesenchymal stromal cells (UC-MSCs are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed-vessel dissociation following enzymatic digestion which reduces contamination risk and manipulation time. The new method produced >10 times more cells per cm of UC than our previous method. When biographical variables were compared, more UC-MSCs per gram were isolated after vaginal birth compared to Caesarian-section births, an unexpected result. UC-MSCs were expanded in medium enriched with 2%, 5%, or 10% pooled human platelet lysate (HPL eliminating the xenogeneic serum components. When the HPL concentrations were compared, media supplemented with 10% HPL had the highest growth rate, smallest cells, and the most viable cells at passage. UC-MSCs grown in 10% HPL had surface marker expression typical of MSCs, high colony forming efficiency, and could undergo trilineage differentiation. The new protocol standardizes manufacturing of UC-MSCs and enables clinical translation.

  16. Selection for resistance to Verticillium wilt caused by race 2 isolates of Verticillium dahliae in accessions of lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Verticillium wilt of lettuce caused by Verticillium dahliae can cause severe economic damage to lettuce producers. The pathogen exists as two races (races 1 and 2) in lettuce, and complete resistance to race 1 is known. Resistance to race 2 isolates has not been reported, and production of race 1 re...

  17. The role of solvent on selective hydrogenation of conjugated and isolated C dbnd C of Citral (3,7-dimethyl 2,6-octadienal) - a self-consistent reaction field study

    Science.gov (United States)

    Chatterjee, Abhijit; Chatterjee, Maya; Ikushima, Yutaka; Mizukami, Fujio

    2004-09-01

    Self-consistent reaction field study was performed to rationalize the selective formation of 3,7-dimethyloctanal by hydrogenation of citral (3,7-dimethyl 2,6-octadienal) exclusively in supercritical carbon dioxide medium. We have used density functional theory (DFT) to calculate and compare the reactivity of the atoms present in the reactant molecule for a range of solvent with variable dielectric constant. Solvent with low dielectric constant was found to be reducing the electrophilicity of the sites present and hence enhances the selectivity of an isolated and conjugated C dbnd C bond-breaking phenomenon. In contrast to that, solvents with high dielectric constant prefer to hydrogenate the conjugated C dbnd C. The geometric and electronic aspects were compared to propose a plausible explanation for the unprecedented selectivity observed in supercritical carbon dioxide medium.

  18. Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

    Science.gov (United States)

    Richel, D J; Johnsen, H E; Canon, J; Guillaume, T; Schaafsma, M R; Schenkeveld, C; Hansen, S W; McNiece, I; Gringeri, A J; Briddell, R; Ewen, C; Davies, R; Freeman, J; Miltenyi, S; Symann, M

    2000-02-01

    The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

  19. Histidine-rich protein 2 (pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

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    Giselle Maria Rachid Viana

    Full Text Available More than 80% of available malaria rapid diagnostic tests (RDTs are based on the detection of histidine-rich protein-2 (PfHRP2 for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3, are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2% of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients. In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples to 50.9% (30 out of 59 samples. In Bolivia, only one of 25 samples (4% tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

  20. Histidine-rich protein 2 (pfhrp2) and pfhrp3 gene deletions in Plasmodium falciparum isolates from select sites in Brazil and Bolivia.

    Science.gov (United States)

    Rachid Viana, Giselle Maria; Akinyi Okoth, Sheila; Silva-Flannery, Luciana; Lima Barbosa, Danielle Regina; Macedo de Oliveira, Alexandre; Goldman, Ira F; Morton, Lindsay C; Huber, Curtis; Anez, Arletta; Dantas Machado, Ricardo Luiz; Aranha Camargo, Luís Marcelo; Costa Negreiros do Valle, Suiane; Marins Póvoa, Marinete; Udhayakumar, Venkatachalam; Barnwell, John W

    2017-01-01

    More than 80% of available malaria rapid diagnostic tests (RDTs) are based on the detection of histidine-rich protein-2 (PfHRP2) for diagnosis of Plasmodium falciparum malaria. Recent studies have shown the genes that code for this protein and its paralog, histidine-rich protein-3 (PfHRP3), are absent in parasites from the Peruvian Amazon Basin. Lack of PfHRP2 protein through deletion of the pfhrp2 gene leads to false-negative RDT results for P. falciparum. We have evaluated the extent of pfhrp2 and pfhrp3 gene deletions in a convenience sample of 198 isolates from six sites in three states across the Brazilian Amazon Basin (Acre, Rondonia and Para) and 25 isolates from two sites in Bolivia collected at different times between 2010 and 2012. Pfhrp2 and pfhrp3 gene and their flanking genes on chromosomes 7 and 13, respectively, were amplified from 198 blood specimens collected in Brazil. In Brazil, the isolates collected in Acre state, located in the western part of the Brazilian Amazon, had the highest percentage of deletions for pfhrp2 25 (31.2%) of 79, while among those collected in Rondonia, the prevalence of pfhrp2 gene deletion was only 3.3% (2 out of 60 patients). In isolates from Para state, all parasites were pfhrp2-positive. In contrast, we detected high proportions of isolates from all 3 states that were pfhrp3-negative ranging from 18.3% (11 out of 60 samples) to 50.9% (30 out of 59 samples). In Bolivia, only one of 25 samples (4%) tested had deleted pfhrp2 gene, while 68% (17 out of 25 samples) were pfhrp3-negative. Among the isolates tested, P. falciparum pfhrp2 gene deletions were present mainly in those from Acre State in the Brazilian Amazon. These results indicate it is important to reconsider the use of PfHRP2-based RDTs in the western region of the Brazilian Amazon and to implement appropriate surveillance systems to monitor pfhrp2 gene deletions in this and other parts of the Amazon region.

  1. Mass Spectrometry-Based Metabolomics of Agave Sap (Agave salmiana after Its Inoculation with Microorganisms Isolated from Agave Sap Concentrate Selected to Enhance Anticancer Activity

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    Luis M. Figueroa

    2017-11-01

    Full Text Available Saponins have been correlated with the reduction of cancer cell growth and the apoptotic effect of agave sap concentrate. Empirical observations of this artisanal Mexican food have shown that fermentation occurs after agave sap is concentrated, but little is known about the microorganisms that survive after cooking, or their effects on saponins and other metabolites. The aim of this study was to evaluate the changes in metabolites found in agave (A. salmiana sap after its fermentation with microorganisms isolated from agave sap concentrate, and demonstrate its potential use to enhance anticancer activity. Microorganisms were isolated by dilution plating and identified by 16S rRNA analysis. Isolates were used to ferment agave sap, and their corresponding butanolic extracts were compared with those that enhanced the cytotoxic activity on colon (Caco-2 and liver (Hep-G2 cancer cells. Metabolite changes were investigated by mass spectrometry-based metabolomics. Among 69 isolated microorganisms, the actinomycetes Arthrobacter globiformis and Gordonia sp. were used to analyze the metabolites, along with bioactivity changes. From the 939 ions that were mainly responsible for variation among fermented samples at 48 h, 96 h, and 192 h, four were correlated to anticancer activity. It was shown that magueyoside B, a kammogenin glycoside, was found at higher intensities in the samples fermented with Gordonia sp. that reduced Hep-G2 viability better than controls. These findings showed that microorganisms from agave sap concentrate change agave sap metabolites such as saponins. Butanolic extracts obtained after agave sap fermentation with Arthrobacter globiformis or Gordonia sp. increased the cancer cell growth inhibitory effect on colon or liver cancer cells, respectively.

  2. Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra.

    Science.gov (United States)

    Ständker, L; Harvey, A L; Fürst, S; Mathes, I; Forssmann, W G; Escalona de Motta, G; Béress, L

    2012-09-15

    An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. A new semi-selective medium for the isolation of Xanthomonas axonopodis pv. dieffenbachiae, the etiological agent of anthurium bacterial blight.

    Science.gov (United States)

    Laurent, P; Chabirand, A; Jouen, E; Robène-Soustrade, I; Gagnevin, L; Hostachy, B; Pruvost, O

    2009-08-01

    Xanthomonas axonopodis pv. dieffenbachiae causes anthurium blight, which is regarded as the most threatening disease for the anthurium industry worldwide. The bacterium is listed as a quarantine pathogen in several regions, including Europe. We evaluated the use of Neomycin-Cephalexin-Trimethoprime-pirMecillinam 4 (NCTM4) medium for its isolation. A total of 104 bacterial strains were inoculated onto NCTM4 and on the previously published Cellobiose-Starch (CS) and Esculin-Trehalose (ET) media. The strain collection included: the anthurium blight pathogen, Xanthomonas strains, for which false positive results are known to occur using serological identification-tests; other bacterial pathogens of anthurium; and representatives of bacteria that are commonly present in the anthurium phyllosphere. Media were evaluated following the ISO 16140 protocol for the validation of alternative methods. Growth of the anthurium blight pathogen was better on NCTM4 and ET media than on CS. NCTM4 provided a better repeatability. It also displayed a lower rate of false positive and false negative results when the pathogen was isolated from plant extracts. This study will lead to improved isolation protocols of the anthurium blight in official procedures. NCTM4 medium could also favourably be used in studies, which aim to further understanding of the biology and epidemiology of this pathogen.

  4. Application and comparison of four selected procedures for the isolation of cell-wall material from the skin of grapes cv. Monastrell

    Energy Technology Data Exchange (ETDEWEB)

    Apolinar-Valiente, R., E-mail: tokay04@hotmail.com [Departamento de Tecnologia de Alimentos, Nutricion y Bromatologia, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia (Spain); Romero-Cascales, I., E-mail: miromero@um.es [Departamento de Tecnologia de Alimentos, Nutricion y Bromatologia, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia (Spain); Lopez-Roca, J.M., E-mail: jmlroca@um.es [Departamento de Tecnologia de Alimentos, Nutricion y Bromatologia, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia (Spain); Gomez-Plaza, E., E-mail: encarnag@um.es [Departamento de Tecnologia de Alimentos, Nutricion y Bromatologia, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia (Spain); Ros-Garcia, J.M., E-mail: jmros@um.es [Departamento de Tecnologia de Alimentos, Nutricion y Bromatologia, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30100 Murcia (Spain)

    2010-02-15

    In order to choose an appropriate cell-wall material (CWM) isolation procedure in grapes cv. Monastrell, four different standard procedures have been tested, and a comparison made of the amount of cell-wall material obtained, its composition and morphology. The CWM was isolated as the 70% ethanol insoluble residue (de Vries method), as the absolute ethanol insoluble residue filtered sequentially through nylon mesh (Nunan method), as the insoluble residue in sodium deoxycholate-phenol-acetic acid-water (Selvendran method) and as the N-[2-hydroxyethyl]-piperazine-N'-2-ethanesulfonic acid (HEPES) insoluble residue (Vidal method). All extractions were done in triplicate and the efficiency of the extractive procedure established. Carbohydrates, proteins, and phenolic compounds were analysed, as the main constituents of CWM. The morphology of the isolated CWM was visualized by scanning electron microscopy (SEM). The Selvendran method had the highest efficiency, while the Nunan method had the lower one. Regarding the carbohydrates composition, the four different CWM were rich in uronic acids and glucose, together with varying amounts of arabinose, xylose, mannose and galactose. The Selvendran method had the lower value of total carbohydrates and the CWM shows more plasmatic membrane impurities in SEM images. The chemical results of the Vidal and de Vries methods were quite similar, but the Vidal method was more time consuming than the de Vries method. According to the results, the de Vries method was chosen to produce a representative cell-wall material fraction from Monastrell grapes skin.

  5. Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land)

    Science.gov (United States)

    Siebert, J.; Hirsch, P.; Friedmann, E. I. (Principal Investigator)

    1988-01-01

    Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys). From these, 15 coccoid strains were chosen for more detailed investigation. They were characterized by morphological, physiological and chemotaxonomical properties. All isolates were Gram-positive, catalase-positive and nonmotile. Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M. roseus, M. agilis) or Deinococcus. In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios. Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C. Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples. Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem. These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.

  6. A novel nontoxic alkyl-phospholipid with selective antitumor activity, plasmanyl-(N-acyl)-ethanolamine (PNAE), isolated from degenerating chick embryonal tissues and from an anticancer biopreparation cACPL.

    Science.gov (United States)

    Kára, J; Borovicka, M; Liebl, V; Smolíková, J; Ubik, K

    1986-01-01

    A novel alkyl-phospholipid with selective antitumor activity was isolated from an anticancer biopreparation cACPL (crude anticancer phospholipids) and from tissues of degenerating chick embryos. The alkyl-phospholipid was isolated and purified by chromatographic methods using silicic acid column chromatography and thin layer chromatography. The chemical structure of the alkyl-phospholipid was characterized by thin-layer chromatographic analysis of the degradation products after enzymatic digestion with phospholipase C from Bacillus cereus and with phospholipase D, by two-dimensional thin-layer chromatography, infrared spectrum, and mass spectrometric analysis. The alkyl-phospholipid was identified as 1-O-alkyl-2-acyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine, i.e. plasmanyl-(N-acyl)-ethanolamine (PNAE), the main molecular species being 1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)-ethanol-amine. PNAE exhibits a selective cytolytic effect on human tumor cells HEp-2, HeLa and T24 in tissue cultures at the concentration of 25 micrograms PNAE per ml and 66-98% inhibition of DNA synthesis in the human tumor cells at concentration as low as 2.5 micrograms/ml, but it does not inhibit at a 50-fold higher concentration the DNA synthesis and normal growth of human fibroblasts (cell line LEP). PNAE represents the main biologically active antitumor component of the cACPL biopreparation and exhibits a significant antitumor effect in vivo in B10/An mice bearing Mc11 fibrosarcoma. Possible molecular mechanism of the selective antitumor activity of PNAE is discussed, involving the selective disturbance of phospholipid metabolism in tumor cells, leading to progressive destruction of tumor cell membranes. The fact that PNAE is nontoxic and selectively active against tumor cells at nanomolar concentrations in vitro as well as in vivo, indicates the possibility of its clinical use. PNAE and cACPL biopreparation might provide a very useful new tool for human anticancer

  7. Atomic force microscopic comparison of remineralization with casein-phosphopeptide amorphous calcium phosphate paste, acidulated phosphate fluoride gel and iron supplement in primary and permanent teeth: An in-vitro study.

    Science.gov (United States)

    Agrawal, Nikita; Shashikiran, N D; Singla, Shilpy; Ravi, K S; Kulkarni, Vinaya Kumar

    2014-01-01

    Demineralization of tooth by erosion is caused by frequent contact between the tooth surface and acids present in soft drinks. The present study objective was to evaluate the remineralization potential of casein-phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste, 1.23% acidulated phosphate fluoride (APF) gel and iron supplement on dental erosion by soft drinks in human primary and permanent enamel using atomic force microscopy (AFM). Specimens were made from extracted 15 primary and 15 permanent teeth which were randomly divided into three treatment groups: CPP-ACP paste, APF gel and iron supplement. AFM was used for baseline readings followed by demineralization and remineralization cycle. Almost all group of samples showed remineralization that is a reduction in surface roughness which was higher with CPP-ACP paste. Statistical analysis was performed using by one-way ANOVA and Mann-Whitney U-test with P paste is effective on preventing dental erosion from soft drinks.

  8. SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa Stably Transfected with SPARC.

    Directory of Open Access Journals (Sweden)

    Andrea Slusser-Nore

    Full Text Available This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3 and cadmium (Cd(+2-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2-and As(+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice.Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3-and Cd(+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF. Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres.It was shown that the As(+3-and Cd(+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3-and Cd(+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells.Tumor initiating cells isolated from SPARC-transfected As(+3-and Cd(+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

  9. Revalorization of selected municipal solid wastes as new precursors of "green" nanocellulose via a novel one-pot isolation system: A source perspective.

    Science.gov (United States)

    Chen, You Wei; Lee, Hwei Voon

    2017-08-30

    In the present work, four types of newly chosen municipal solid wastes (Panax ginseng, spent tea residue, waste cotton cloth, and old corrugated cardboard) were studied as the promising sources for nanocellulose, which has efficiently re-engineered the structure of waste products into highly valuable nanocellulose materials. The nanocellulose was produced directly via a facile one-pot oxidative hydrolysis process by using H2O2/Cr(NO3)3 solution as the bleaching agent and hydrolysis medium under acidic condition. The isolated nanocellulose products were well-characterized in terms of chemical composition, product yield, morphological structure and thermal properties. The study has found that the crystallinity index of the obtained nanocellulose products were significantly higher (62.2-83.6%) than that of its starting material due to the successive elimination of lignin, hemicellulose and amorphous regions of cellulose, which were in good agreement with the FTIR analysis. The evidence of the successful production of nanocellulose was given by TEM observation which has revealed the fibril widths were ranging from 15.6 to 46.2nm, with high cellulose content (>90%), depending on the cellulosic origin. The physicochemical properties of processed samples have confirmed that the isolation of high purity nanocellulose materials from different daily spent products is possible. The comparative study can help to provide a deep insight on the possibility of revalorizing the municipal solid wastes into nanocellulose via the simple and versatile one-pot isolation system, which has high potential to be used in commercial applications for sustainable development. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens

    DEFF Research Database (Denmark)

    Abdul Momin, Muhd Haziq F; Bean, David C; Hendriksen, Rene S.

    2017-01-01

    . Using CHROMagar COL-APSE, Gram-negative pathogens (n=71) with intrinsic (n=8) or acquired COL (n=63) resistance were recovered with 100 % specificity down to the lower limit of detection of 101 colony-forming units (c.f.u.). The growth on SuperPolymyxin was similar, but notably weaker for COL...... for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1...

  11. Large-scale isolation of CD34+ cells using the Amgen cell selection device results in high levels of purity and recovery.

    Science.gov (United States)

    McNiece, I; Briddell, R; Stoney, G; Kern, B; Zilm, K; Recktenwald, D; Miltenyi, S

    1997-02-01

    The Amgen Cell Selection Device (ACSD) is a fully automated system based on the research scale magnetic-activated cell separation (MACS) system (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) for the selection of CD34+ cells. Leukapheresis products (LP) (n = 30) from normal donors mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) were selected with the ACSD to evaluate the performance of this system. The starting LP contained a median of 0.51% CD34+ cells (range 0.21%-1.54%) and a median WBC count of 3.0 x 10(10) (range 1-4.7 x 10(10) cells). After selection on the ACSD a mean purity of 91.5% +/- 0.6% CD34+ cells was obtained, with a median purity of 95.5% CD34+ cells. A median of 98 x 10(6) total CD34+ cells were recovered postselection, with a range of 31-323 x 10(6) cells collected from the LP. This represented a mean recovery of 81.7% +/- 6% of CD34+ cells and a median of 78% compared with starting CD34+ cell numbers in the LP. FACS analysis of the selected products demonstrated a 4-5 log depletion of T cell subsets, including CD3, CD4, CD8, and CD56 subsets. These data demonstrate the high performance obtained with the ACSD resulting in a final product of greater than 90% purity of CD34+ cells. CD34+ cells selected with the ACSD represent an ideal product for clinical applications, such as tumor cell purging, T cell depletion for allogeneic transplant, ex vivo expansion, and gene therapy.

  12. Determination of selected pesticides in water samples adjacent to agricultural fields and removal of organophosphorus insecticide chlorpyrifos using soil bacterial isolates

    Science.gov (United States)

    Hossain, M. S.; Chowdhury, M. Alamgir Zaman; Pramanik, Md. Kamruzzaman; Rahman, M. A.; Fakhruddin, A. N. M.; Alam, M. Khorshed

    2015-06-01

    The use of pesticide for crops leads to serious environmental pollution, therefore, it is essential to monitor and develop approaches to remove pesticide from contaminated environment. In this study, water samples were collected to monitor pesticide residues, and degradation of chlorpyrifos was also performed using soil bacteria. Identification of pesticide residues and determination of their levels were performed by high-performance liquid chromatography with photodiode array detector. Among 12 samples, 10 samples were found contaminated with pesticides. Chlorpyrifos was detected in four tested samples and concentrations ranged from 3.27 to 9.31 μg/l whereas fenitrothion ranging from (Below Detection Limit, removal of 50 mg/l chlorpyrifos. Chlorpyrifos degradation rates were found maximum by all three isolates at 2nd day of incubation for both tested concentrations. The results of the present study suggest the need for regular monitoring of pesticide residues in water, to protect the aquatic environment. Chlorpyrifos degrading bacterial isolates can be used to clean up environmental samples contaminated with the organophosphate pesticides.

  13. LCMS-QTOF Determination of Lentinan-Like β-D-Glucan Content Isolated by Hot Water and Alkaline Solution from Tiger’s Milk Mushroom, Termite Mushroom, and Selected Local Market Mushrooms

    Directory of Open Access Journals (Sweden)

    Nor Azreen Mohd Jamil

    2013-01-01

    Full Text Available Lentinan, 1152 Dalton β-D-glucan found in Shiitake Mushroom (Lentinus edodes, has been claimed to have anticancer and immunomodulatory activity. Several extraction methods have been used by researchers to isolate Lentinan including hot water and alkaline solution (1.25 M NaOH. In this study, hot water and alkaline solution (1.25 M NaOH were used to extract the Lentinan-like β-D-glucan (1151 Dalton from Tiger’s Milk Mushroom, Termite Mushroom, and selected local market mushrooms. The isolated Lentinan-like β-D-glucan from both hot water and alkaline solution was analyzed by LCMS-QTOF. Commercial Lentinan standard from Lentinus edodes was used as a reference. The results showed significant differences on chromatogram patterns of Lentinan-like β-D-glucan between both extracts. The peak of Lentinan-like β-D-glucan was only found in isolated polysaccharide glucan of hot water extracts. The isolated polysaccharide glucans from Tiger’s Milk Mushroom and Termite Mushroom were found to have 0.74±0.12 μg/mg and 0.53±0.07 μg/mg Lentinan-like β-D-glucan. Button Mushroom, Shiitake Mushroom, and Oyster Mushroom showed the presence of Lentinan-like β-D-glucan at 16.16±4.15 μg/mg, 0.22±0.04, and 0.10±0.01 μg/mg, respectively.

  14. Isolation, characterization and identification of actinomycetes from ...

    African Journals Online (AJOL)

    STORAGESEVER

    antimicrobial test using selected phytopathogens as test strains and it was observed that 3, 25, 35 and. 37 of the isolates showed antagonistic reaction with ... Six of the most promising isolates were selected and identified using their 16S rRNA sequence. ..... isolated from a Brazilian tropical forest soil. World J. Microbiol.

  15. Evaluation of Antimicrobial Potency and Phytochemical Screening of Persea americana Leaf Extracts against Selected Bacterial and Fungal Isolates of Clinical Importance

    OpenAIRE

    O. E. Ajayi; S. I. Awala; O. T. Olalekan; O. A. Alabi

    2017-01-01

    Aim: The quest for novel bioactive from natural sources informed the evaluation of the antimicrobial alongside the phytochemical composition of leaf extracts of Persea americana obtained from Akure, Ondo State, Nigeria. Study Design: This study assessed the prospective antimicrobial efficacy of Persia americana against selected clinically relevant bacteria and fungi. Place and Duration of Study: The study was conducted between April and September, 2015 at the Microbiology Laboratory o...

  16. Isolation of a novel retinoic acid-responsive gene by selection of genomic fragments derived from CpG-island-enriched DNA.

    Science.gov (United States)

    Shago, M; Giguére, V

    1996-08-01

    One of the primary goals in transcription factor research is the elucidation of the genetic networks controlled by a factor or by members of a family of closely related factors. The pleiotropic effects of retinoic acid (PA) in the developing and adult animal are mediated by ligand-inducible transcription factors (RA receptors [RARs] and retinoid X receptors [RXRs]) that belong to the superfamily of nuclear receptors. Regulatory regions of PA effector genes contain RAR and RXR binding sites (RAR elements [RAREs] and RXR elements [RXREs]) that generally consist of direct or everted repeats of the core half-site motif, (A/G)G(G/T)TCA. In order to identify novel genes regulated by RA, we devised a selection strategy based on the premise that regulatory regions of a large number of housekeeping and tissue-specific genes are embodied within CpG island DNA. In this method, referred to as CpG-selected and amplified binding, fragments derived from the CpG island fraction of the murine genome are selected by a gel mobility shift assay using in vitro-transcribed and -translated RXR-RAR. Multiple rounds of selection coupled with amplification of the fragments by PCR enabled us to clone a population of CG-rich fragments of which approximately one-fifth contained consensus RAREs or RXREs. Twelve genomic fragments containing novel response elements are described, and the transcription unit associated with one of them, NN-84AG, was characterized in detail. The mouse NN-84AG transcript is upregulated by RA in F9 embryonal carcinoma cells and is homologous to an expressed sequence tag (EST41159) derived from a human infant brain cDNA library. Cloning of the murine NN8-4AG genomic sequence places the RXRE in the proximity of the transcription initiation sites of the gene. Although sequence analysis indicates that the EST41159 gene product is novel, a region of amino acid identity with sequences of a yeast polypeptide of, as yet, unknown function and the Drosophila trithorax protein

  17. Isolation and characterisation of selected germander diterpenoids from authenticated Teucrium chamaedrys and T. canadense by HPLC, HPLC-mS and NMR.

    Science.gov (United States)

    Sundaresan, P Ramnathan; Slavoff, Sarah A; Grundel, Erich; White, Kevin D; Mazzola, Eugene; Koblenz, Daniel; Rader, Jeanne I

    2006-01-01

    Teucrium species, such as germander, are rich in neo-clerodane diterpenoids and have been used in traditional folk medicine for their stimulant, diuretic, antipyretic and antiseptic properties. However, the furano neo-clerodane diterpenoids present in germander have been implicated in the in vivo hepatotoxicity of this botanical. In this study, authenticated germander (Teucrium chamaedrys L. and Teucrium canadense L.) was used as the source material. Methanol extracts of powdered plant mate rial were prepared and analysed by HPLC using Synergi Max-RP columns with monitoring at 220 nm. Limited amounts of teucrin A and other diterpenoid standards were analysed on a Synergi Max-RP column in order to determine their retention times and to generate calibration curves. The same standards were subjected to concurrent mass spectral analysis. Teucrin A and diterpenoids such as dihydroteugin, teuflin, teuflidin and teucvidin were tentatively identified in the plant extracts by HPLC-MS and 1H-NMR experiments. For the isolation of teucrium diterpenoids on a semipreparative scale, a solid-phase extraction method was developed for the first time using styrene divinylbenzene and strata-X sorbents for teucrin A and teuflin, respectively. Semi-preparative HPLC of the methanol extract of the powdered aerial parts of Teucrium plants was carried out on a semipreparative Synergi Max-RP column with photodiode array detection in order to confirm the identities of some diterpenoids by HPLC-MS and NMR.

  18. Social isolation

    National Research Council Canada - National Science Library

    Cacioppo, John T; Hawkley, Louise C; Norman, Greg J; Berntson, Gary G

    2011-01-01

    ..., reproduce, and care for offspring sufficiently long that they too reproduced. Social isolation represents a lens through which to investigate these behavioral, neural, hormonal, cellular, and genetic mechanisms...

  19. Phosphorylation of the pyruvate dehydrogenase complex isolated from Ascaris suum

    Energy Technology Data Exchange (ETDEWEB)

    Thissen, J.; Komuniecki, R.

    1987-05-01

    The pyruvate dehydrogenase complex (PDC) from body wall muscle of the porcine nematode, Ascaris suum, plays a pivotal role in anaerobic mitochondrial metabolism. As in mammalian mitochondria, PDC activity is inhibited by the phosphorylation of the ..cap alpha..PDH subunit, catalyzed by an associated PDH/sub a/ kinase. However, in contrast to PDC's isolated from all other eukaryotic sources, phosphorylation decreases the mobility of the ..cap alpha..PDH subunit on SDS-PAGE and permits the separation of the phosphorylated and nonphosphorylated ..cap alpha..PDH's. Phosphorylation and the inactivation of the Ascaris PDC correspond directly, and the additional phosphorylation that occurs after complete inactivation in mammalian PDC's is not observed. The purified ascarid PDC incorporates 10 nmoles /sup 32/P/mg P. Autoradiography of the radiolabeled PDC separated by SDS-PAGE yields a band which corresponds to the phosphorylated ..cap alpha..PDH and a second, faint band which is present only during the first three minutes of PDC inactivation, intermediate between the phosphorylated and nonphosphorylated ..cap alpha..PDH subunit. Tryptic digests of the /sup 32/P-PDC yields one major phosphopeptide, when separated by HPLC, and its amino acid sequence currently is being determined.

  20. Endothelium-dependent relaxant responses to selective 5-HT(1B/1D) receptor agonists in the isolated middle cerebral artery of the rat

    DEFF Research Database (Denmark)

    Hansen-Schwartz, Jacob; Løvland Hoel, Natalie; Nilsson, Elisabeth

    2003-01-01

    response to 5-HT and triptans. Using the vessel bath technique, MCA segments were mounted on two metal wires. The relaxant responses to sumatriptan could not be reproduced using this model; instead, weak contractile responses (6 +/- 3% of submaximal contractile capacity) were observed. The difference...... perfused. Luminally added 5- hydroxytryptamine (5-HT), sumatriptan and rizatriptan induced maximal dilatations of 22 +/- 4, 10 +/- 2 and 13 +/- 5%, respectively, compared to the resting diameter. The relaxant effect of sumatriptan was blocked by the 5- HT(1B/1D) receptor selective antagonist GR 55562 (10......(-6)M). The use of N(omega)-nitro-L-arginine and charybdotoxin revealed that the dilatation involved both nitric oxide and endothelially derived hyperpolarising factor. Thus, the earlier demonstrated expression of 5-HT(1B/1D) immunoreactivity in the endothelium may well translate into a relaxant...

  1. Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: an efficient tool in the control of natural steroid hormones abuse in cattle.

    Science.gov (United States)

    Doué, Mickael; Bichon, Emmanuelle; Dervilly-Pinel, Gaud; Pichon, Valérie; Chapuis-Hugon, Florence; Lesellier, Eric; West, Caroline; Monteau, Fabrice; Le Bizec, Bruno

    2012-12-28

    The use of anabolic substances to promote growth in livestock is prohibited within the European Union as laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopic deviation measurements allow to control natural steroid hormones abuse. In both cases, urine is often selected as the most relevant matrix and, due to its relatively high content of potential interferents, its preparation before analysis is considered as a key step. In this context, the use of a selective sorbent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized based on 17β-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively. Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroid purification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative supercritical fluid chromatography with a commercial MIP as stationary phase (SFC-MIP). Parameters for both approaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandrolone and boldenone. The MISPE protocol developed for screening purposes allowed satisfactory recoveries (upper 65% for the 12 target steroids) with sufficient purification for gas chromatography-mass spectrometry (GC-MS) analysis. For confirmatory purposes, the use of isotopic ratio mass spectrometry (IRMS) requires a higher degree of purity of the target compounds, which can be reached by the SFC-MIP protocol with three steps less compared to the official and current method. Purity, concentration and absence of isotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone, estradiol, androstenedione, and boldenone) allowed the measurement of (13)C/(12)C isotopic ratios of corresponding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy. Copyright © 2012 Elsevier B.V. All rights

  2. Genetic characterization of an extended-spectrum AmpC cephalosporinase with hydrolysing activity against fourth-generation cephalosporins in a clinical isolate of Enterobacter aerogenes selected in vivo.

    Science.gov (United States)

    Rodríguez-Martínez, Jose M; Fernández-Echauri, Pedro; Fernández-Cuenca, Felipe; Diaz de Alba, Paula; Briales, Alejandra; Pascual, Alvaro

    2012-01-01

    Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we characterize a new AmpC variant presenting a broadened substrate activity towards fourth-generation cephalosporins, selected in vivo following cefepime treatment for Enterobacter aerogenes. Two consecutive clonally related isolates of E. aerogenes were evaluated. Screening for ESAC production was performed using plates containing 200 mg/L cloxacillin. MICs were determined by microdilution (CLSI guidelines). bla(AmpC) genes were cloned into a pCR-Blunt II-TOPO vector and expressed in Escherichia coli. The ampC genes were cloned into vector pGEX-6P-1 for protein purification. Isolate Ea595 was resistant to two fourth-generation cephalosporins, cefepime and cefpirome; using plates containing cloxacillin, susceptibility to ceftazidime and cefepime was restored, suggesting overproduction of the ESAC β-lactamase. Sequencing identified a new AmpC β-lactamase variant presenting one amino acid substitution, Val291Gly, inside the H-10 helix. Recombinant plasmids harbouring this ESAC β-lactamase conferred a broadened resistance profile to cefepime and cefpirome, with resistance levels increasing from 16- to 32-fold in E. coli. AmpC-Ea595 hydrolysed ceftazidime, cefepime and cefpirome at high levels, presenting a lower K(m) and enabling us to classify the enzyme as an ESAC. Homology modelling suggested that the size of the active site could have increased. We characterized an ESAC β-lactamase selected in vivo and conferring a high level of resistance to fourth-generation cephalosporins in E. aerogenes. The broadened spectrum was caused by a new modification to the H-10 helix, which modified the active site.

  3. β-Caryophyllene, a Compound Isolated from the Biblical Balm of Gilead (Commiphora gileadensis), Is a Selective Apoptosis Inducer for Tumor Cell Lines

    Science.gov (United States)

    Amiel, Eitan; Ofir, Rivka; Dudai, Nativ; Soloway, Elaine; Rabinsky, Tatiana; Rachmilevitch, Shimon

    2012-01-01

    The biblical balm of Gilead (Commiphora gileadensis) was investigated in this study for anticancerous activity against tumor cell lines. The results obtained from ethanol-based extracts and from essential oils indicated that β-caryophyllene (trans-(1R,9S)-8-methylene-4,11,11-trimethylbicyclo[7.2.0]undec-4-ene) is a key component in essential oils extracted from the balm of Gilead. β-Caryophyllene can be found in spice blends, citrus flavors, soaps, detergents, creams, and lotions, as well as in a variety of food and beverage products, and it is known for its anti-inflammatory, local anaesthetic, and antifungal properties. It is also a potent cytotoxic compound over a wide range of cell lines. In the current paper, we found that Commiphora gileadensis stem extracts and essential oil have an antiproliferative proapoptotic effect against tumor cells and not against normal cells. β-caryophyllene caused a potent induction of apoptosis accompanied by DNA ladder and caspase-3 catalytic activity in tumor cell lines. In summary, we showed that C. gileadensis stems contain an apoptosis inducer that acts, in a selective manner, against tumor cell lines and not against normal cells. PMID:22567036

  4. β-Caryophyllene, a Compound Isolated from the Biblical Balm of Gilead (Commiphora gileadensis, Is a Selective Apoptosis Inducer for Tumor Cell Lines

    Directory of Open Access Journals (Sweden)

    Eitan Amiel

    2012-01-01

    Full Text Available The biblical balm of Gilead (Commiphora gileadensis was investigated in this study for anticancerous activity against tumor cell lines. The results obtained from ethanol-based extracts and from essential oils indicated that β-caryophyllene (trans-(1R,9S-8-methylene-4,11,11-trimethylbicyclo[7.2.0]undec-4-ene is a key component in essential oils extracted from the balm of Gilead. β-Caryophyllene can be found in spice blends, citrus flavors, soaps, detergents, creams, and lotions, as well as in a variety of food and beverage products, and it is known for its anti-inflammatory, local anaesthetic, and antifungal properties. It is also a potent cytotoxic compound over a wide range of cell lines. In the current paper, we found that Commiphora gileadensis stem extracts and essential oil have an antiproliferative proapoptotic effect against tumor cells and not against normal cells. β-caryophyllene caused a potent induction of apoptosis accompanied by DNA ladder and caspase-3 catalytic activity in tumor cell lines. In summary, we showed that C. gileadensis stems contain an apoptosis inducer that acts, in a selective manner, against tumor cell lines and not against normal cells.

  5. Enhanced production of polygalacturonase in solid-state fermentation: selection of the process conditions, isolation and partial characterization of the enzyme.

    Science.gov (United States)

    Zaslona, Halina; Trusek-Holownia, Anna

    2015-01-01

    Polygalacturonase (PG) production by Penicillium chrysogenum during solid-state fermentation was accompanied by decomposition of orange peels. A leaching procedure was developed through the selection of solvent, time and intensity of stirring. A maximum PG activity was observed after 48 h peel inoculation. Further cultivation decreased the enzyme activity significantly, up to 60% of the maximum PG activity. During fermentation, a rapid acidification of the solid medium which inhibited the pectinolytic enzyme, was observed. Buffering agents with different pH values and different ionic strengths were examined to identify the most suitable medium to avoid this problem. Buffer addition counteracted acidification and enhanced active protein production, which was observed for all of the applied pH values (6.5-8.0) of the buffering agent. The most satisfactory results were obtained when using the highest pH at 8.0. The protein content and PG activity increased from 3.5 mg/g and 1.09 U/g to 7.7 mg/g and 7.11 U/g during cultivation, with uncontrolled and pH-controlled medium, respectively. Measurements at wide pH and temperature ranges indicated an optimum for PG activity at pH 5.0 and 43°C; however, high thermal stability corresponded to lower temperatures, and a temperature of 37°C is thus recommended. Under these conditions, the operational stability was determined to be t1/2=570 h.

  6. Isolated Aortitis

    Science.gov (United States)

    ... among others. In some cases, biologics like anti-tumor necrosis factor drugs such as infliximab or rituximab have been used. Isolated aortitis occurring with retroperitoneal fibrosis has been treated with tamoxifen, an anti- ...

  7. Speciation analysis of calcium, iron, and zinc in casein phosphopeptide fractions from toddler milk-based formula by anion exchange and reversed-phase high-performance liquid chromatography-mass spectrometry/flame atomic-absorption spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Miquel, Esther; Alegria, Amparo; Barbera, Reyes; Farre, Rosaura [University of Valencia, Nutrition and Food Chemistry, Faculty of Pharmacy, Burjassot, Valencia (Spain)

    2005-03-01

    Casein phosphopeptides (CPP) are phosphorylated casein-derived peptides that can be released by in-vitro or in-vivo enzymatic hydrolysis of {alpha}{sub s1}-casein, {alpha}{sub s2}-casein, and {beta}-casein (CN). Many of these peptides contain a highly polar acidic sequence of three phosphoseryl groups followed by two glutamic acid residues. These domains are binding sites for minerals such as calcium, iron, and zinc and play an important role in mineral bioavailability. The aim of this study was speciation analysis of calcium, iron, and zinc in CPP fractions from the soluble fraction of a toddler milk-based formula. Methods for CPP separation by anion-exchange high-performance liquid chromatography (AE-HPLC) were combined with CPP identification by reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry and determination of the calcium, iron, zinc, and phosphorus content of the fractions obtained by AE-HPLC. Calcium and phosphorus were detected in all the analyzed AE-HPLC fractions. Calcium and zinc could be bound to CPP derived from {alpha}{sub s1}-CN and {alpha}{sub s2}-CN in fraction 3. Iron could be bound to CPP in fraction 4 in which {beta}-CN(15-34)4P was present with the cluster sequence S(P)S(P)S(P)EE. The results obtained prove the different distribution of calcium, iron, and zinc in heterogeneous CPP fractions. (orig.)

  8. Atomic force microscopic comparison of remineralization with casein-phosphopeptide amorphous calcium phosphate paste, acidulated phosphate fluoride gel and iron supplement in primary and permanent teeth: An in-vitro study

    Directory of Open Access Journals (Sweden)

    Nikita Agrawal

    2014-01-01

    Full Text Available Context: Demineralization of tooth by erosion is caused by frequent contact between the tooth surface and acids present in soft drinks. Aim: The present study objective was to evaluate the remineralization potential of casein-phosphopeptide-amorphous calcium phosphate (CPP-ACP paste, 1.23% acidulated phosphate fluoride (APF gel and iron supplement on dental erosion by soft drinks in human primary and permanent enamel using atomic force microscopy (AFM. Materials and Methods: Specimens were made from extracted 15 primary and 15 permanent teeth which were randomly divided into three treatment groups: CPP-ACP paste, APF gel and iron supplement. AFM was used for baseline readings followed by demineralization and remineralization cycle. Results and Statistics: Almost all group of samples showed remineralization that is a reduction in surface roughness which was higher with CPP-ACP paste. Statistical analysis was performed using by one-way ANOVA and Mann-Whitney U-test with P < 0.05. Conclusions: It can be concluded that the application of CPP-ACP paste is effective on preventing dental erosion from soft drinks.

  9. Selection of Beauveria bassiana and Metarhizium anisopliae isolates for the control of Blissus antillus (Hemiptera: Lygaeidae Seleção de isolados Beauveria Bassiana e Metarhizium Anisopliae para o controle de Blissus Antillus

    Directory of Open Access Journals (Sweden)

    Richard Ian Samuels

    2004-06-01

    Full Text Available Chinch bugs of the genus Blissus (Hemiptera: Lygaeidae are serious pests of maize, wheat, sorghum and various grasses. This study is the first to screen isolates of Beauveria bassiana and Metarhizium anisopliae against 4th instar and adult Blissus antillus aiming to develop a biological control program for this important pasture pest. Ten fungal isolates were initially screened and three isolates were chosen for further investigation. To determine virulence, insects were inoculated by immersion in concentrations of 5 ´ 10(8 conidia mL-1. Mortality was evaluated for 10 days. B. bassiana ARSEF 792 was the most virulent isolate to both nymphs and adults, causing 53 and 78% infection, respectively, and values for LT50 of 7.8 and 5.0 days, respectively. Germination studies were carried out to confirm viability and determine speed of germination as a pathogenicity factor. The production of conidia on the cadavers of insects infected with the three selected isolates was determined. The production of conidia on rice media was also evaluated. B. bassiana CG 24 produced the highest number of conidia on insects cadavers (14.9 ´ 10(7 conidia per insect and also on rice media (10.6 ´ 10(9 conidia per g.Percevejos do gênero Blissus (Hemiptera: Lygeidae são pragas importantes das plantações de milho, trigo e sorgo e de vários tipos de gramíneos forrageiros. Esse estudo é o primeiro a amostrar isolados de Beauveria bassiana e Metarhizium anisopliae contra aos 4(0 instares e adultos de Blissus antillus com a finalidade de desenvolver um programa de controle biológico dessa importante praga de pastagens. Dez isolados foram inicialmente testados e três foram escolhidos para maiores investigações. Para determinar a virulência, os insetos foram inoculados por imersão em suspensões de conídios a uma concentração de 5 ´ 10(8 conídios mL-1. Mortalidade foi avaliada por 10 dias. B. bassiana ARSEF 792 foi o isolado mais virulento para as ninfas e os

  10. Isolating Triggered Star Formation

    Energy Technology Data Exchange (ETDEWEB)

    Barton, Elizabeth J.; Arnold, Jacob A.; /UC, Irvine; Zentner, Andrew R.; /KICP, Chicago /Chicago U., EFI; Bullock, James S.; /UC, Irvine; Wechsler, Risa H.; /KIPAC, Menlo

    2007-09-12

    Galaxy pairs provide a potentially powerful means of studying triggered star formation from galaxy interactions. We use a large cosmological N-body simulation coupled with a well-tested semi-analytic substructure model to demonstrate that the majority of galaxies in close pairs reside within cluster or group-size halos and therefore represent a biased population, poorly suited for direct comparison to 'field' galaxies. Thus, the frequent observation that some types of galaxies in pairs have redder colors than 'field' galaxies is primarily a selection effect. We use our simulations to devise a means to select galaxy pairs that are isolated in their dark matter halos with respect to other massive subhalos (N= 2 halos) and to select a control sample of isolated galaxies (N= 1 halos) for comparison. We then apply these selection criteria to a volume-limited subset of the 2dF Galaxy Redshift Survey with M{sub B,j} {le} -19 and obtain the first clean measure of the typical fraction of galaxies affected by triggered star formation and the average elevation in the star formation rate. We find that 24% (30.5 %) of these L* and sub-L* galaxies in isolated 50 (30) h{sup -1} kpc pairs exhibit star formation that is boosted by a factor of {approx}> 5 above their average past value, while only 10% of isolated galaxies in the control sample show this level of enhancement. Thus, 14% (20 %) of the galaxies in these close pairs show clear triggered star formation. Our orbit models suggest that 12% (16%) of 50 (30) h{sup -1} kpc close pairs that are isolated according to our definition have had a close ({le} 30 h{sup -1} kpc) pass within the last Gyr. Thus, the data are broadly consistent with a scenario in which most or all close passes of isolated pairs result in triggered star formation. The isolation criteria we develop provide a means to constrain star formation and feedback prescriptions in hydrodynamic simulations and a very general method of understanding

  11. The selective isolation of the uterine oestradiol-receptor complex by binding to oligo(dT)-cellulose. The mediation of an essential activator in the transformation of cytosol receptor.

    Science.gov (United States)

    Thrower, S; Hall, C; Lim, L; Davison, A N

    1976-11-15

    The [3H]oestradiol-receptor complex was selectively isolated from rat uterus cytosol by column chromatography on oligo(dT)-cellulose. Optimal conditions are described for the binding of the complex to oligo(dT)-cellulose, which is shown to be similar to its binding to DNA-cellulose. The cytosol complex has an apparent mol. wt. of 50,000-60,000 in high salt concentrations, as determined by Sephadex G-100 chromatography. This corresponds to the 4S cytoplasmic oestradiol receptor. In binding to oligo(dT)-cellulose the receptor is transformed into a form with an apparent mol.wt. of 100,000-120,000, corresponding to the 5S nuclear receptor complex. This transformation mimics the conversion in vivo of the cytoplasmic oestradiol receptor into the nuclear form. The binding of the complex to oligo(dT)-cellulose as a 5S nuclear form is unequivocally demonstrated to require the mediation of an activating present in the cytosol. The requirement for an activating factor is discussed in relation to reports that nuclear binding of the oestradiol-receptor complex is not dictated solely by the availability of the cytoplasmic oestradiol receptor.

  12. Improvement of the butanol production selectivity and butanol to acetone ratio (B:A) by addition of electron carriers in the batch culture of a new local isolate of Clostridium acetobutylicum YM1.

    Science.gov (United States)

    Nasser Al-Shorgani, Najeeb Kaid; Kalil, Mohd Sahaid; Wan Yusoff, Wan Mohtar; Shukor, Hafiza; Hamid, Aidil Abdul

    2015-12-01

    Improvement in the butanol production selectivity or enhanced butanol:acetone ratio (B:A) is desirable in acetone-butanol-ethanol (ABE) fermentation by Clostridium strains. In this study, artificial electron carriers were added to the fermentation medium of a new isolate of Clostridium acetobutylicum YM1 in order to improve the butanol yield and B:A ratio. The results revealed that medium supplementation with electron carriers changed the metabolism flux of electron and carbon in ABE fermentation by YM1. A decrease in acetone production, which subsequently improved the B:A ratio, was observed. Further improvement in the butanol production and B:A ratios were obtained when the fermentation medium was supplemented with butyric acid. The maximum butanol production (18.20 ± 1.38 g/L) was gained when a combination of methyl red and butyric acid was added. Although the addition of benzyl viologen (0.1 mM) and butyric acid resulted in high a B:A ratio of 16:1 (800% increment compared with the conventional 2:1 ratio), the addition of benzyl viologen to the culture after 4 h resulted in the production of 18.05 g/L butanol. Manipulating the metabolic flux to butanol through the addition of electron carriers could become an alternative strategy to achieve higher butanol productivity and improve the B:A ratio. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Selección de cepas de rizobios aisladas de ecosistemas ganaderos de Canadá, inoculadas en trigo (Triticum aestivum, L.. Selection of rhizobium strains isolated from livestock ecosystems of Canada, inoculated to wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    C.J Bécquer

    2007-06-01

    Full Text Available Se efectuó un experimento de campo con el objetivo de seleccionar cepas de rizobios inoculadas en trigo (Triticum aestivum, L., var. Cuba-204. Se evaluaron varios indicadoress agroproductivos de la planta, tales como: peso seco aéreo, longitud del tallo, rendimiento de granos, peso de 1000 granos y rendimiento de nitrógeno. Como criterio de selección de los mejores tratamientos se consideraron los valores estadísticamente superiores al control fertilizado. Se utilizaron cuatro cepas de referencia, pertenecientes a diferentes géneros y especies de rizobios, y 10 cepas nativas, pertenecientes al género Sinorhizobium, que fueron aisladas de raíces de leguminosas (Melilotus y Medicago, adaptadas a ecosistemas ganaderos de Alberta, Canadá. Una de las cepas fue aislada de leguminosas adaptadas a suelos contaminados con hidrocarburos de esa misma zona geográfica. Las cepas crecieron en LMA y resuspendidas en CLM hasta lograr una UFC de 10(6 _ 10(8 cél/mL. Se utilizaron métodos estándar para la inoculación de cereales. Se aplicó un diseño experimental de bloques completamente aleatorizado, con 16 tratamientos y cuatro réplicas. Uno de los tratamientos se fertilizó con 150 kg/ha (NH4NO3. Se utilizó análisis de varianza. Las diferencias entre medias fueron halladas por la prueba LSD de Fisher (pA field experiment was carried out in order to select rhizobial strains inoculated to wheat (Triticum aestivum, L., var. Cuba-204. Several parameters of agricultural significance were assessed, such as: aerial dry weight, length of stems, grain yield, weight of 1 000 grains and nitrogen yield. As selection criterion of the best treatments, the statistically higher values compared to the fertilized control were considered. Four reference strains, belonging to several rhizobial genus and species were used, as well as ten native strains, belonging to Sinorhizobium that were isolated from roots of legumes (Melilotus and Medicago, adapted to Canadian

  14. Schizosaccharomyces isolation method

    Directory of Open Access Journals (Sweden)

    Benito Santiago

    2014-01-01

    Full Text Available This study discusses the optimization of a selective and differential medium which would facilitate the isolation of Schizosaccharomyces (a genus with a low incidence compared to other microorganisms to select individuals from this genus for industrial purposes, especially in light of the recent recommendation of the use of yeasts from this genus in the wine industry by the International Organisation of Vine and Wine, or to detect the presence of such yeasts, for those many authors who consider them food spoilers. To this end, we studied various selective differential agents based on the main physiological characteristics of these species, such as their high resistances to high concentrations of sugar, sulfur dioxide, sorbic acid, benzoic acid, acetic acid or malo ethanolic fermentation. This selective medium is based on the genus resistance to the antibiotic actidione and its high resistance to inhibitory agents such as benzoic acid. Malic acid was used as a differential factor due to the ability of this genus to metabolise it to ethanol, which allows detecting of the degradation of this compound. Lastly, the medium was successfully used to isolate strains of Schizosaccharomyces pombe from honey and honeycombs.

  15. Employment of High-Performance Thin-Layer Chromatography for the Quantification of Oleuropein in Olive Leaves and the Selection of a Suitable Solvent System for Its Isolation with Centrifugal Partition Chromatography.

    Science.gov (United States)

    Boka, Vasiliki-Ioanna; Argyropoulou, Aikaterini; Gikas, Evangelos; Angelis, Apostolis; Aligiannis, Nektarios; Skaltsounis, Alexios-Leandros

    2015-11-01

    A high-performance thin-layer chromatographic methodology was developed and validated for the isolation and quantitative determination of oleuropein in two extracts of Olea europaea leaves. OLE_A was a crude acetone extract, while OLE_AA was its defatted residue. Initially, high-performance thin-layer chromatography was employed for the purification process of oleuropein with fast centrifugal partition chromatography, replacing high-performance liquid-chromatography, in the stage of the determination of the distribution coefficient and the retention volume. A densitometric method was developed for the determination of the distribution coefficients, KC = CS/CM. The total concentrations of the target compound in the stationary phase (CS) and in the mobile phase (CM) were calculated by the area measured in the high-performance thin-layer chromatogram. The estimated Kc was also used for the calculation of the retention volume, VR, with a chromatographic retention equation. The obtained data were successfully applied for the purification of oleuropein and the experimental results confirmed the theoretical predictions, indicating that high-performance thin-layer chromatography could be an important counterpart in the phytochemical study of natural products. The isolated oleuropein (purity > 95%) was subsequently used for the estimation of its content in each extract with a simple, sensitive and accurate high-performance thin-layer chromatography method. The best fit calibration curve from 1.0 µg/track to 6.0 µg/track of oleuropein was polynomial and the quantification was achieved by UV detection at λ 240 nm. The method was validated giving rise to an efficient and high-throughput procedure, with the relative standard deviation % of repeatability and intermediate precision not exceeding 4.9% and accuracy between 92% and 98% (recovery rates). Moreover, the method was validated for robustness, limit of quantitation, and limit of detection. The amount of oleuropein for

  16. The Simultaneous Effect of Extended Etching Time and Casein Phosphopeptide-Amorphous Calcium Phosphate containing Paste Application on Shear Bond Strength of Etch-and-rinse Adhesive to Caries-affected Dentin.

    Science.gov (United States)

    Doozandeh, Maryam; Firouzmandi, Maryam; Mirmohammadi, Milad

    2015-10-01

    This study evaluated the simultaneous effect of extended etching time and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) containing paste application on shear bond strength (SBS) of etch-and-rinse adhesive to caries-affected-dentin (CAD). Fifty human third molars were used; 10 normal in group 1 and 40 caries-affected teeth in groups 2 to 5. In the CAD groups, teeth were randomly assigned into four groups (n = 10). In groups 2 and 4, phosphoric acid etching for 15 and 45 seconds was used and in groups 3 and 5, after 15 or 45 seconds etching respectively, CPP-ACP containing paste (MI paste) was applied for 3 minutes. After rinsing, single bond adhesive system and Z250 composite were used for resin composite build-up in all groups. After storage, SBS test was measured at cross head speed of 0.5 mm/min. Data were analyzed using one/two-way ANOVA and Tukey HSD test (α = 0.05). Normal dentin groups showed the highest SBS among different groups. There was a statistically significant difference between different etching times in CAD groups (p adhesive to CAD with or without CPP-ACP pretreated in both etching times. Most of the failure modes were adhesive except in groups 1 and 5 which cohesive failure in composite was also observed. The results of our study indicated that extended etching time up to 45 seconds could enhance the SBS of CAD and approach nearly to SBS of normal dentin. Moreover, MI paste pretreatment had not significant effect on SBS of etch-and-rinse adhesive in CAD. Simultaneous application of extended etching time and CPP-ACP containing paste may be a suggested method in increasing the SBS of adhesive to CAD.

  17. The effects of TAK-875, a selective G protein-coupled receptor 40/free fatty acid 1 agonist, on insulin and glucagon secretion in isolated rat and human islets.

    Science.gov (United States)

    Yashiro, Hiroaki; Tsujihata, Yoshiyuki; Takeuchi, Koji; Hazama, Masatoshi; Johnson, Paul R V; Rorsman, Patrik

    2012-02-01

    G protein-coupled receptor 40 (GPR40)/free fatty acid 1 (FFA1) is a G protein-coupled receptor involved in free fatty acid-induced insulin secretion. To analyze the effect of our novel GPR40/FFA1-selective agonist, [(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate (TAK-875), on insulin and glucagon secretion, we performed hormone secretion assays and measured intracellular Ca²⁺ concentration ([Ca²⁺](i)) in both human and rat islets. Insulin and glucagon secretion were measured in static and dynamic conditions by using groups of isolated rat and human pancreatic islets. [Ca²⁺](i) was recorded by using confocal microscopy. GPR40/FFA1 expression was measured by quantitative polymerase chain reaction. In both human and rat islets, TAK-875 enhanced glucose-induced insulin secretion in a glucose-dependent manner. The stimulatory effect of TAK-875 was similar to that produced by glucagon-like peptide-1 and correlated with the elevation of β-cell [Ca²⁺](i). TAK-875 was without effect on glucagon secretion at both 1 and 16 mM glucose in human islets. These data indicate that GPR40/FFA1 influences mainly insulin secretion in a glucose-dependent manner. The β-cell-specific action of TAK-875 in human islets may represent a therapeutically useful feature that allows plasma glucose control without compromising counter-regulation of glucagon secretion, thus minimizing the risk of hypoglycemia.

  18. Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3.

    Science.gov (United States)

    Flarakos, Caroline Ceailles; Weiskopf, Andrew; Robinson, Matthew; Wang, Guoshun; Vouros, Paul; Sasso, Gino J; Uskokovic, Milan R; Reddy, G Satyanarayana

    2017-03-01

    Analogs of 1α,25-dihydroxyvitamin D3 (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D3 (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs. For example, 1α,25-dihydroxy-16-ene-20-epi-vitamin D3 (S4), but not 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR-106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3-epi-S4, a metabolite of S4 produced through C-3 epimerization pathway. Using GC/MS, ESI-MS and (1)H NMR analysis, we identified all the four LPMs of S4 as 25-hydroxy-16-ene-20-epi-vitamin D3-1-stearate and 25-hydroxy-16-ene-20-epi-vitamin D3-1-oleate and their respective C-3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR-106 cells in which both 1α,25(OH)2-16-ene-20-epi-D3 and 1α,25(OH)2-16-ene-20-epi-3-epi-D3 undergo C-1 esterification into stearic and oleic acid esters. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Modification of calcite crystal morphology by designed phosphopeptides and primary structures and substrate specifities of the cysteine proteases mexicain and chymomexicain

    Science.gov (United States)

    Lian, Zhirui

    In order to better understand the mechanism of biomineralization, we have undertaken to synthesize polypeptide model compounds of well-defined structure that can interact with specific faces of calcite and alter its crystal morphology. These peptides were designed based on the structure of alpha-helical winter flounder antifreeze polypeptide HPLC-6. In these peptides, from one to three of the threonine residues in HPLC-6 were substituted by phosphoserine or phosphotyrosine. CD spectra show that all the peptides have virtually the same alpha-helicity, i.e., about 90% at 4°C and 50% at 25°C. However, only peptides which contain at least two phosphate groups spaced 16.8-A apart can modify the crystal morphology of the calcite. The newly developed surface has been tentatively identified as the (001) basal face. Molecular modeling indicates that the spacing of phosphate groups allows for a good match with crystal lattice ions on the (001) plane. Another peptide, CBP-3D, in which the three threonine residues in HPLC-6 were substituted by aspartic acids, appears to bind only to {104} rhombohedral faces of calcite. These experiments suggest that conformation and orientation of the binding ligands in the peptide are important factors governing the mutual recognition of crystal surface and proteins. The complete amino acid sequences of the cysteine proteases mexicain and chymomexicain, isolated from the latex of the plant Pileus mexicanus , were determined by Edman degradation of proteolytic fragments. Mexicain and chymomexicain show-high sequence homology to the papain family of cysteine protease. Mexicain and chymomexicain are monomeric polypeptides, with molecular masses of 23,762 Da and 23,694 Da, respectively, and both contain three deduced disulfide bonds. The proteolytic substrate specificities of mexicain and chymomexicain were studied by digesting a series of synthetic peptides and analyzing the fragments by mass spectrometry. The two proteases showed virtually

  20. Probiotic properties of Lactobacillus strains isolated from Tibetan kefir grains

    National Research Council Canada - National Science Library

    Zheng, Yongchen; Lu, Yingli; Wang, Jinfeng; Yang, Longfei; Pan, Chenyu; Huang, Ying

    2013-01-01

    ...) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected...

  1. Sexual selection and natural selection in bird speciation

    OpenAIRE

    Price, T.

    1998-01-01

    The role of sexual selection in speciation is investigated, addressing two main issues. First, how do sexually selected traits become species recognition traits? Theory and empirical evidence suggest that female preferences often do not evolve as a correlated response to evolution of male traits. This implies that, contrary to runaway (Fisherian) models of sexual selection, premating isolation will not arise as an automatic side effect of divergence between populations in sexually selected tr...

  2. Genetic diversity of Escherichia coli isolated from commercial swine ...

    African Journals Online (AJOL)

    PCR) for the analysis of genetic diversity among Escherichia coli strains isolated from commercial swine farms in Sichuan province of China. Thirty four strains of E. coli were selected by selective medium and conventional biochemical test from ...

  3. Porosity, single-phase permeability, and capillary pressure data from preliminary laboratory experiments on selected samples from Marker Bed 139 at the Waste Isolation Pilot Plant. Volume 3 of 3: Appendices C, D, E, and F

    Energy Technology Data Exchange (ETDEWEB)

    Howarth, S.M.; Christian-Frear, T.

    1997-08-01

    This volume contains the mineralogy, porosity, and permeability results from the Marker Bed 139 anhydrite specimens evaluated by TerraTek, Inc. for the Waste Isolation Pilot Plant. This volume also documents the brine recipe used by RE/SPEC, Inc., the parameter package submitted to Performance Assessment based on all the data, and a memo on the mixed Brooks and Corey two-phase characteristic curves.

  4. Assessment of microbial diversity in the rhizosphere of Pinus roxburghii (Sarg.) and bio-inoculant potential of selected pine bacterial isolates for wheat varieties based on culture independent and culture dependent techniques.

    Science.gov (United States)

    Naz, I; Bano, A; Mirza, M S

    2017-10-15

    Evidences are missing regarding compatibility of pine bacteria as bio-inoculants for crops. The diversity and abundance of rhizosphere bacteria of Pinus roxburghii had never been investigated by the simultaneous application of culture dependent and culture independent techniques. Present study was aimed to isolate, characterize, to check the bio-inoculant potential of pine bacteria and to assess rhizosphere bacterial diversity using culture independent advance approaches. Fourty bacteria were isolated from the rhizoplane of P. roxburghii growing in cold climate at high altitude in Murree, morphologically characterized; nine were identified by 16S-rRNA sequence analyses and used in experiment. The diversity and abundance of 16S-rRNA gene and nifH gene in the rhizosphere was assessed by cloning, RFLP analysis, 454 amplicon Pyrosequencing and qPCR. The bacterial isolates significantly improved dry weight of shoot, root, root area, IAA, GA3 contents, no. of grains/plant, weight of grains/plant in wheat var. Chakwal-50 and Fareed-06 under axenic and field conditions. The no. of 16S-rRNA sequences (2979) identified by Pyrosequencing shared similarity with 13 phyla of domains Bacteria and Archaea. The results confirmed the existence of diverse bacteria of agricultural and industrial importance in the rhizosphere and compatibility of rhizoplane bacteria as bio-inoculants for wheat varieties. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  5. Isolation of Salmonella and Shigella species from house flies ...

    African Journals Online (AJOL)

    Salmonella and Shigella species were isolated from House flies (Musca domestica L.) from various sampling sites using selective media. Out of 34 pooled samples Shigella species were isolated in all (100%) of the samples while Salmonella species were isolated in 21 (61.7%) of the samples. The flies pooled from the ...

  6. Clinical bacterial isolates from hospital environment as agents of ...

    African Journals Online (AJOL)

    Bacteria were isolated from the air, floor and patients' surgical wounds in the theatre and surgical ward by using MacConkey agar, Chocolate agar Nutrient agar and Peptone water broth as isolating media. Plasmid sizes and bands of selected twenty (20) of the isolates were determined by electrophoresis analysis to ...

  7. Potency of fibrolytic bacteria isolated from Indonesian sheep's colon ...

    African Journals Online (AJOL)

    Three fibrolytic bacteria were isolated from sheep's colon using cellulose (b), xylan (c) and lignin (d) as selective substrates. The potency of fibrolytic was identified by Subbarao methods. These isolates were then used both in pure and mixed culture with cattle cellulolytic bacteria (a) from the previous research. The isolates ...

  8. Optimization of DNA isolation and PCR protocol for RAPD analysis ...

    African Journals Online (AJOL)

    hope&shola

    of DNA isolation and PCR conditions for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular ... The technique is ideal for isolation of DNA from different plant species and the DNA isolated was used for ..... showed a reading of between 1.6 to 1.7 after calculating the 260/280 nm ...

  9. Isolation and characterization of human rhinovirus antigenic variants

    Energy Technology Data Exchange (ETDEWEB)

    Watson, D.G.

    1985-01-01

    Isolation of antigenic variants of human rhinovirus types 2, 14, and 17 was attempted by plaquing untreated virus (P-isolates), selecting variants in the presence of homologous antiserum (C-isolates), and by selecting variants in the presence of antibody following 5-fluorouracil mutagenesis (M-isolates). All viruses were triple-plaque purified and purity neutralization tested prior to isolate selection. Based on a fourfold reduction in neutralizing antibody titer to homologous antiserum, no antigenic variation was found in P-isolates from the three serotypes examined. Antigenic variants of all three serotypes could be isolated by the antiserum selection method (C-isolates). However, antigenic variants of RV17 were isolated at a much higher frequency and showed a larger degree of variation than those of RV2 and RV14. At least two of the variants selected, RV17 (C301) and RV2 (M803), failed to be neutralized by the known 89 rhinovirus antiserum. SDS-polyacrylamide gel electrophoresis of (/sup 35/S) methionine-labelled virion polypeptides revealed that each serotype had a characteristic pattern and that selected RV2 and RV17 isolates had patterns identical to those of the prototype strains. By isoelectric focusing an antigenic variant of RV2 was shown to contain altered virion polypeptides VP1 and VP2 whereas two RV17 antigenic variants demonstrated alterations only in the VP1 polypeptide.

  10. Individual Influence on Model Selection

    Science.gov (United States)

    Sterba, Sonya K.; Pek, Jolynn

    2012-01-01

    Researchers in psychology are increasingly using model selection strategies to decide among competing models, rather than evaluating the fit of a given model in isolation. However, such interest in model selection outpaces an awareness that one or a few cases can have disproportionate impact on the model ranking. Though case influence on the fit…

  11. Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase and molecular sequencing of selected strains

    Directory of Open Access Journals (Sweden)

    Mauro Cintra Giudice

    2012-09-01

    Full Text Available A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum. The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

  12. Avaliação da eficiência de três ágares seletivos no isolamento de Listeria monocytogenes Evaluation of the efficiency of three selective agars for L. monocytogenes isolation

    Directory of Open Access Journals (Sweden)

    Denize Aparecida Rodrigues

    2003-12-01

    Full Text Available A variedade de protocolos existentes para a pesquisa de Listeria sp em alimentos e outras amostras é muito grande, o que dificulta a escolha daquele que possa apresentar melhores resultados. Os protocolos recomendados nas metodologias tradicionais indicam vários caldos de pré-enriquecimento e enriquecimento seletivo, assim como meios de isolamento seletivo. O presente estudo teve por finalidade comparar a eficiência dos ágares cloreto de lítio-feniletanol-moxalactam (LPM, PALCAM (PAL e do ágar hemolítico ceftazidima-cloreto de lítio (HCLA no isolamento de L. monocytogenes a partir de diferentes tipos de amostras colhidas em uma linha de processamento de "nuggets" congelados de frango. Um total de 413 amostras foi examinado. As amostras foram primeiramente enriquecidas em caldo Half Fraser, seguido do enriquecimento em caldo BLEB. Alíquotas deste enriquecimento foram semeadas por esgotamento em placas contendo os meios LPM, PAL e HCLA. Colônias típicas foram selecionadas e submetidas à identificação bioquímica. O meio HCLA permitiu o isolamento de L. monocytogenes em 60,1% do total de amostras examinadas, enquanto PAL e LPM permitiram o isolamento em aproximadamente 47,9% das amostras. O desempenho do HCLA foi estatisticamente diferente dos demais meios (pThere is a variety of protocols for Listeria spp detection in foods and other sources. Therefore, the decision on which method to be used becomes very difficult. Conventional isolation methodologies indicate different pre-enrichment and enrichment broths as well as different isolation media. The comparison between the efficiency of PALCAM (PAL, lithium chloride-phenylethanol-moxalactam (LPM and haemolitic ceftazidime-lithium chloride (HCLA agars was carried out. A total of 413 samples from different sources, collected from a chicken nuggets processing plant, was pre-enriched in Half Fraser broth followed by enrichment in BLEB. They were streaked onto LPM, PAL and HCLA plates and

  13. Characterization and selection of Bacillus thuringiensis isolates effective against Sitophilus oryzae Caracterização e seleção de isolados de Bacillus thuringiensis efetivos contra Sitophilus oryzae

    Directory of Open Access Journals (Sweden)

    Najara da Silva

    2010-08-01

    Full Text Available The entomopathogenic bacterium Bacillus thuringiensis is a control agent with toxic and environmental characteristics that allows the control of pest insects according to the Integrate Pest Management (IPM precepts. In order to find new strains, potentially toxic to Sitophilus oryzae L. 1763 (Coleoptera: Curculinidae, 1.073 strains of B. thuringiensis from parts of Brazil were used. Genetic material was extracted with InstaGene Matrix kit, used for the amplification of sequences in Polymerase chain reaction (PCR, and viewed in 1.5% agarose gel. The gene cry35Ba class was represented by 60 B. thuringiensis isolates (5.6%, which were then subjected to bioassays with S. oryzae larvae. Among the isolates studied, four caused more than 50% mortality in pathogenicity tests, and the isolates 544 and 622 were the most virulent, as determined by CL50 estimates. The four toxic isolates had spherical, bi-pyramidal and cuboid crystals, and a 44-kDa protein was found in sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE, which coded for the product of cry35Ba genes. These data demonstrate the potential of B. thuringiensis for the management of S. oryzae larvae.A bactéria entomopatogênica Bacillus thuringiensis (Bt é um agente de controle com características tóxicas e ambientais que permitem o controle de insetos-praga de acordo com as premissas do Manejo integrado de pragas (MIP. Com o objetivo de buscar novas linhagens potencialmente tóxicas para Sitophilus oryzae L. 1763 (Coleoptera: Curculinidae, caracterizaram-se molecularmente 1,073 isolados de B. thuringiensis de regiões do Brasil. O material genético foi extraído através do kit InstaGene Matrix, utilizado para a amplificação das seqüências através da técnica de Polymerase chain reaction PCR, sendo os resultados visualizados em gel de agarose 1,5%. A classe do gene cry35Ba foi representada por 60 isolados (5,6% de Bt, os quais foram submetidos a bioensaio com larvas

  14. Occurrence of molds on laminated paperboard for aseptic packaging, selection of the most hydrogen peroxide- and heat-resistant isolates and determination of their thermal death kinetics in sterile distilled water.

    Science.gov (United States)

    Delgado, Denise Aparecida; de Souza Sant'ana, Anderson; de Massaguer, Pilar Rodriguez

    2012-07-01

    This study aimed at enumerating molds (heat-labile and heat-resistant) on the surface of paperboard material to be filled with tomato pulps through an aseptic system and at determining the most heat- and hydrogen peroxide-resistant strains. A total of 118 samples of laminated paperboard before filling were collected, being 68 before and 50 after the hydrogen peroxide bath. Seven molds, including heat-resistant strains (Penicillium variotii and Talaromyces flavus) with counts ranging between 0.71 and 1.02 CFU/cm(2) were isolated. P. variotii was more resistant to hydrogen peroxide than T. flavus and was inactivated after heating at 85 °C/15 min. When exposed to 35 % hydrogen peroxide at 25 °C, T. flavus (F5E2) and N. fischeri (control) were less resistant than P. variotti (F1A1). P. citrinum (F7E2) was shown to be as resistant as P. variotti. The D values (the time to cause one logarithmic cycle reduction in a microbial population at a determined temperature) for spores of P. variotii (F1A1) and N. fischeri (control) with 4 months of age at 85 and 90 °C were 3.9 and 4.5 min, respectively. Although the contamination of packages was low, the presence of heat- and chemical-resistant molds may be of concern for package sterility and product stability during shelf-life. To our knowledge, this is the first report that focuses on the isolation of molds, including heat-resistant ones, contaminating paperboard packaging material and on estimating their resistance to the chemical and physical processes used for packaging sterilization.

  15. Isolated patellofemoral osteoarthritis.

    NARCIS (Netherlands)

    Jonbergen, H.P. van; Poolman, R.W.; Kampen, A. van

    2010-01-01

    BACKGROUND AND PURPOSE: The optimal treatment for isolated patellofemoral osteoarthritis is unclear at present. We systematically reviewed the highest level of available evidence on the nonoperative and operative treatment of isolated patellofemoral osteoarthritis to develop an evidenced-based

  16. Fractionation of T cell subsets on Ig anti-Ig columns: isolation of helper T cells from nonresponder mice, demonstration of antigen-specific T suppressor cells, and selection of CD-3 negative variants of Jurkat T cells

    DEFF Research Database (Denmark)

    Rubin, B; Geisler, C; Kuhlmann, J

    1989-01-01

    In the present experiments we have explored the possibilities of a modified immunoadsorbent technique to select for (1) mutagenized T cell receptor (Tcr) negative variants of Jurkat T lymphoma cells and (2) purified CD-4+ or CD-8+ T lymphocytes. The basic principle was to make large numbers...... of immunoglobulin (Ig) negative T cells Ig+ by T cell subset-specific monoclonal antibodies (mAb), and to select such cells on Ig anti-Ig columns. Our results demonstrated that Thy-1+, Fc receptor positive, antigen-specific T cells regulate the immune response in mice nonresponders to pork insulin......." The most important finding is the demonstration of antigen-specific Thy-1+, CD-8+, and Fc receptor+ T suppressor cell that apparently react with antigen in a non-major histocompatibility complex-restricted manner....

  17. The selective A-type K+ current blocker Tx3-1 isolated from the Phoneutria nigriventer venom enhances memory of naïve and Aβ(25-35)-treated mice.

    Science.gov (United States)

    Gomes, Guilherme M; Dalmolin, Gerusa D; Cordeiro, Marta do Nascimento; Gomez, Marcus V; Ferreira, Juliano; Rubin, Maribel A

    2013-12-15

    Potassium channels regulate many neuronal functions, including neuronal excitability and synaptic plasticity, contributing, by these means, to mnemonic processes. In particular, A-type K(+) currents (IA) play a key role in hippocampal synaptic plasticity. Therefore, we evaluated the effect of the peptidic toxin Tx3-1, a selective blocker of IA currents, extracted from the venom of the spider Phoneutria nigriventer, on memory of mice. Administration of Tx3-1 (i.c.v., 300 pmol/site) enhanced both short- and long-term memory consolidation of mice tested in the novel object recognition task. In comparison, 4-aminopyridine (4-AP; i.c.v., 30-300 pmol/site), a non-selective K(+) channel blocker did not alter long-term memory and caused toxic side effects such as circling, freezing and tonic-clonic seizures. Moreover, Tx3-1 (i.c.v., 10-100 pmol/site) restored memory of Aβ25-35-injected mice, and exhibited a higher potency to improve memory of Aβ25-35-injected mice when compared to control group. These results show the effect of the selective blocker of IA currents Tx3-1 in both short- and long-term memory retention and in memory impairment caused by Aβ25-35, reinforcing the role of IA in physiological and pathological memory processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Postzygotic Isolation Evolves before Prezygotic Isolation between Fresh and Saltwater Populations of the Rainwater Killifish, Lucania parva

    Directory of Open Access Journals (Sweden)

    Genevieve M. Kozak

    2012-01-01

    Full Text Available Divergent natural selection has the potential to drive the evolution of reproductive isolation. The euryhaline killifish Lucania parva has stable populations in both fresh water and salt water. Lucania parva and its sister species, the freshwater L. goodei, are isolated by both prezygotic and postzygotic barriers. To further test whether adaptation to salinity has led to the evolution of these isolating barriers, we tested for incipient reproductive isolation within L. parva by crossing freshwater and saltwater populations. We found no evidence for prezygotic isolation, but reduced hybrid survival indicated that postzygotic isolation existed between L. parva populations. Therefore, postzygotic isolation evolved before prezygotic isolation in these ecologically divergent populations. Previous work on these species raised eggs with methylene blue, which acts as a fungicide. We found this fungicide distorts the pattern of postzygotic isolation by increasing fresh water survival in L. parva, masking species/population differences, and underestimating hybrid inviability.

  19. Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

    Science.gov (United States)

    Mohammed, S I; Johnson, D E; Aronson, A I

    1996-11-01

    Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.

  20. Porosity, single-phase permeability, and capillary pressure data from preliminary laboratory experiments on selected samples from Marker Bed 139 at the Waste Isolation Pilot Plant. Volume 1 of 3: Main report, appendix A

    Energy Technology Data Exchange (ETDEWEB)

    Howarth, S.M.; Christian-Frear, T.

    1997-08-01

    Three groups of core samples from Marker Bed 139 of the Salado Formation at the Waste Isolation Pilot Plant (WIPP) were analyzed to provide data to support the development of numerical models used to predict the long-term hydrologic and structural response of the WIPP repository. These laboratory experiments, part of the FY93 Experimental Scoping Activities of the Salado Two-Phase Flow Laboratory Program, were designed to (1) generate WIPP-specific porosity and single-phase permeability data, (2) provide information needed to design and implement planned tests to measure two-phase flow properties, including threshold pressure, capillary pressure, and relative permeability, and (3) evaluate the suitability of using analog correlations for the Salado Formation to assess the long-term performance of the WIPP. This report contains a description of the boreholes core samples, the core preparation techniques used, sample sizes, testing procedures, test conditions, and results of porosity and single-phase permeability tests performed at three laboratories: TerraTek, Inc. (Salt Lake City, UT), RE/SPEC, Inc. (Rapid City, SD), and Core Laboratories-Special Core Analysis Laboratory (Carrollton, TX) for Rock Physics Associates. In addition, this report contains the only WIPP-specific two-phase-flow capillary-pressure data for twelve core samples. The WIPP-specific data generated in this laboratory study and in WIPP field-test programs and information from suitable analogs will form the basis for specification of single- and two-phase flow parameters for anhydrite markers beds for WIPP performance assessment calculations.

  1. Impact of V2 mutations on escape from a potent neutralizing anti-V3 monoclonal antibody during in vitro selection of a primary human immunodeficiency virus type 1 isolate.

    Science.gov (United States)

    Shibata, Junji; Yoshimura, Kazuhisa; Honda, Akiko; Koito, Atsushi; Murakami, Toshio; Matsushita, Shuzo

    2007-04-01

    KD-247, a humanized monoclonal antibody to an epitope of gp120-V3 tip, has potent cross-neutralizing activity against subtype B primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess how KD-247 escape mutants can be generated, we induced escape variants by exposing bulked primary R5 virus, MOKW, to increasing concentrations of KD-247 in vitro. In the presence of relatively low concentrations of KD-247, viruses with two amino acid mutations (R166K/D167N) in V2 expanded, and under high KD-247 pressure, a V3 tip substitution (P313L) emerged in addition to the V2 mutations. However, a virus with a V2 175P mutation dominated during passaging in the absence of KD-247. Using domain swapping analysis, we demonstrated that the V2 mutations and the P313L mutation in V3 contribute to partial and complete resistance phenotypes against KD-247, respectively. To identify the V2 mutation responsible for the resistance to KD-247, we constructed pseudoviruses with single or double amino acid mutations in V2 and measured their sensitivity to neutralization. Interestingly, the neutralization phenotypes were switched, so that amino acid residue 175 (Pro or Leu) located in the center of V2 was exchanged, indicating that the amino acid at position 175 has a crucial role, dramatically changing the Env oligomeric state on the membrane surface and affecting the neutralization phenotype against not only anti-V3 antibody but also recombinant soluble CD4. These data suggested that HIV-1 can escape from anti-V3 antibody attack by changing the conformation of the functional envelope oligomer by acquiring mutations in the V2 region in environments with relatively low antibody concentrations.

  2. Fasciola hepatica: Histology of the Reproductive Organs and Differential Effects of Triclabendazole on Drug-Sensitive and Drug-Resistant Fluke Isolates and on Flukes from Selected Field Cases

    Directory of Open Access Journals (Sweden)

    Robert Hanna

    2015-06-01

    Full Text Available This review summarises the findings of a series of studies in which the histological changes, induced in the reproductive system of Fasciola hepatica following treatment of the ovine host with the anthelmintic triclabendazole (TCBZ, were examined. A detailed description of the normal macroscopic arrangement and histological features of the testes, ovary, vitelline tissue, Mehlis’ gland and uterus is provided to aid recognition of the drug-induced lesions, and to provide a basic model to inform similar toxicological studies on F. hepatica in the future. The production of spermatozoa and egg components represents the main energy consuming activity of the adult fluke. Thus the reproductive organs, with their high turnover of cells and secretory products, are uniquely sensitive to metabolic inhibition and sub-cellular disorganisation induced by extraneous toxic compounds. The flukes chosen for study were derived from TCBZ-sensitive (TCBZ-S and TCBZ-resistant (TCBZ-R isolates, the status of which had previously been proven in controlled clinical trials. For comparison, flukes collected from flocks where TCBZ resistance had been diagnosed by coprological methods, and from a dairy farm with no history of TCBZ use, were also examined. The macroscopic arrangement of the reproductive system in flukes was studied using catechol/carmine stained whole mounts, and the histology of the main organs was examined using conventional haematoxylin-eosin stained sections. Validation of apoptosis in the fluke sections was carried out using an in situ hybridisation method designed to label endonuclease-induced DNA strand breaks. In TCBZ-S flukes exposed to TCBZ metabolites for 24–96 h in vivo, but not in TCBZ-R flukes, those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles, were found to display progressive loss of cell content. This was due to apparent failure of cell division to keep pace with expulsion of the mature or

  3. Organic-inorganic hybrid silica monolith based immobilized titanium ion affinity chromatography column for analysis of mitochondrial phosphoproteome.

    Science.gov (United States)

    Hou, Chunyan; Ma, Junfeng; Tao, Dingyin; Shan, Yichu; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2010-08-06

    A novel kind of immobilized metal affinity chromatography (IMAC) column based on organic-inorganic hybrid silica monolith has been developed. The monolithic support was prepared in a 250 microm i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, amine groups were functionalized by glutaraldehyde, and then activated with (aminomethyl) phosphonic acid, followed by Ti(4+) chelation. By such a hybrid silica monolithic Ti(4+)-IMAC column, 15 phosphopeptides were effectively isolated from the digest mixture of alpha-casein and BSA with the molar ratio as low as 1:200, illustrating its superior selectivity. With a synthetic phosphorylated peptide, YKVPQLEIVPNSpAEER, as the sample, the loading capacity and recovery of the Ti(4+)-IMAC monolithic column were measured to be 1.4 micromol/mL and 69%, respectively. Such an IMAC monolithic column was further applied to enrich phosphopeptides from rat liver mitochondria. In total, 224 unique phosphopeptides, corresponding to 148 phosphoprotein groups, were identified by duplicate nanoRPLC-LTQ MS/MS/MS runs with a false-positive rate of less than 1% at the peptide level. These results demonstrate that the hybrid silica monolith based Ti(4+)-IMAC column might provide a promising tool for large-scale phosphopeptide enrichment, facilitating the in-depth understanding of the biological functions of phosphoproteomes.

  4. Trypsin functionalization and zirconia coating of mesoporous silica nanotubes for matrix-assisted laser desorption/ionization mass spectrometry analysis of phosphoprotein.

    Science.gov (United States)

    Zhang, Xiaoli; Wang, Fei; Xia, Yan

    2013-09-06

    Trypsin functionalized mesoporous silica nanotubes bioreactor (TEMSN) and zirconia layer coated mesoporous silica nanotubes (ZrO2-MSN) were developed to deal with the long in-solution digestion time of phosphoprotein and detection difficulty of phosphorylated peptides, respectively. Trypsin was immobilized on the mesoporous silica nanotubes via epoxy group and TEMSN were used as a bioreactor for digestion of α-casein within 3min. ZrO2-MSN were performed to enrich phosphopeptides selectively from in-solution digested peptide mixture of β-casein to demonstrate that ZrO2-MSN possessed remarkable selectivity for phosphorylated peptides even at 100/1 molar ratio of BSA/β-casein. The selective ability of ZrO2-MSN was also investigated in comparison to ZrO2 nanoparticles (ZrO2 NP). Moreover, phosphorylated peptides at the femtomole (2.5fmol) level can also be detected with high S/N (signal-to-noise) ratio. Phosphopeptides enriched from TEMSN-bioreactor digested peptide mixture of α-casein was also performed to evaluate the cooperative performance of TEMSN and ZrO2-MSN platform. The experimental results indicated that TEMSN-bioreactor digestion changed the distribution of relative abundance of phosphopeptides and improved the relative intensity of partial phosphopeptides. This analytical strategy has also been applied to the identification of phosphopeptides isolated from non-fat bovine milk and got a comparable results compared with other materials cited from the literature. By matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), TEMSN and ZrO2-MSN were combined together for the rapid and comprehensive analysis of phosphoprotein. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Isolation of site-specific anharmonicities of individual water molecules in the I-·(H2O)2 complex using tag-free, isotopomer selective IR-IR double resonance

    Science.gov (United States)

    Yang, Nan; Duong, Chinh H.; Kelleher, Patrick J.; Johnson, Mark A.; McCoy, Anne B.

    2017-12-01

    We reveal the microscopic mechanics of iodide ion microhydration by recording the isotopomer-selective vibrational spectra of the I-·(H2O)·(D2O), I-·(HOD)·(D2O), and I-·(DOH)·(H2O) isotopologues using a new class of ion spectrometer that is optimized to carry out two-color, IR-IR photodissociation in a variety of pump-probe schemes. Using one of these, we record the linear absorption spectrum of a cryogenically cooled cluster without the use of a messenger ;tag;. In another protocol, we reveal the spectra of individual H2O and D2O molecules embedded in each of the two possible binding sites in the iodide dihydrate, as well as the bands due to individual OH and OD groups in each of the four local binding environments. Finally, we demonstrate how temperature dependent isotopic scrambling among the spectral features can be used to monitor the onset of large amplitude motion, heretofore inferred from changes in the envelope of the OH stretching vibrational manifold.

  6. Selective Fatalism.

    OpenAIRE

    Sunstein, Cass R

    1998-01-01

    Human beings are selectively fatalistic. Some risks appear as "background noise," whereas other, quantitatively identical risks cause enormous concern. This essay explores the reasons for selective fatalism and possible legal responses. Sometimes selective fatalism is a product of distributional issues, as people focus especially on risks that face particular groups; sometimes people adapt their preferences and beliefs so as to reduce concern with risks that they perceive themselves unable to...

  7. ISOLATES OF CANINE DIS TEMPER VIRUS

    African Journals Online (AJOL)

    Three groups of dogs aged three months each were used in an experiment to assess efficacy of imported Canine distemper vaccine (Ondersteport strain) and measles vaccine in protecting Nigerian dogs against local isolates of Canine distemper virus. Each group consisted of four randomly selected puppies. One group ...

  8. Benchmark selection

    DEFF Research Database (Denmark)

    Hougaard, Jens Leth; Tvede, Mich

    2002-01-01

    Within a production theoretic framework, this paper considers an axiomatic approach to benchmark selection. It is shown that two simple and weak axioms; efficiency and comprehensive monotonicity characterize a natural family of benchmarks which typically becomes unique. Further axioms are added...... in order to obtain a unique selection...

  9. Alternativ isolering i bygninger

    DEFF Research Database (Denmark)

    Andersen, T.; Skibstrup Eriksen, S.

    Denne SBI-meddelelse beskriver resultaterne af en undersøgelse af bygninger, hvor der er anvendt alternativ isolering. Undersøgelsen omfatter termografering og fugtmåling af udvendige bygningsdele isoleret med alternativ isolering. Endvidere rapporteres resultaterne af målinger af indholdet af tu...

  10. Isolation, purification and identification of bacteria from the shoes ...

    African Journals Online (AJOL)

    ... subtilis (5 subspecies), Bacillus spore (3 subspecies), Bacillus anthracis, Staphylococcus aureus, Bacillus amyloliquefaciens and Bacillus thuringiensis. The results may contribute to the selection of efficient antimicrobial agents for children's shoes and insole. Key words: Children's shoes, bacteria, isolation, identification.

  11. Antibacterial Activity of Lactic Acid Bacteria Isolated from Salad ...

    African Journals Online (AJOL)

    , Lactobacillus plantarum, Leuconostoc mesenteroides and Pediococci sp were isolated from the surfaces of five salad vegetables. The cell free supernatants of the selected LAB were able to inhibit the growth of the indicator pathogens used in ...

  12. Isolation and screening of basidiomycetes with high peroxidative activity.

    NARCIS (Netherlands)

    Jong, de E.; Vries, de F.P.; Field, J.A.; Zwan, van der R.P.; Bont, de J.A.M.

    1992-01-01

    Sixty-seven Poly R-478 decolorizing basidiomycetes were isolated with a selective medium (containing hemp (Cannabis sativa) stem wood, guaiacol and benomyl). Several of the new isolates seemed to be promising manganese peroxidase-containing white-rot fungi. Enzyme assays indicated that either

  13. Cellulolytic activities of wild type fungi isolated from decayed wood ...

    African Journals Online (AJOL)

    Prof. Ogunji

    Damaso, M. C. T., Terzi, S. D., Farias, X. A., de Oliveria, C. P., Fraga, M. E. and Couri, S. (2012). Selection of cellulolytic fungi isolated from diverse substrates. Braz. Arch. Biol. Technol.55 (4): 513 – 520. Doolotkeldieva, T. D. and Bobusheva, S. T. (2011). Screening of wild type fungal isolates for cellulolytic activity. Microbiol.

  14. Enrichment and isolation of microbial strains degrading bioplastic ...

    African Journals Online (AJOL)

    Polyvinyl alcohol (PVA) degrading bacterial strains were isolated from various environmental sites rich in plastic wastes by using the enrichment culture technique. Among the various isolated strains, the selected potent PVA degrading bacterial strains were tentatively characterized as Bacillus and Pseudomonas sp.

  15. Isolation of four hydrocarbon effluent-degrading Bacillaceae species ...

    African Journals Online (AJOL)

    Four bacterial strains belonging to the family Bacillaceae were isolated from a polluted site and tested for their efficiency in degrading a refinery effluent highly polluted with hydrocarbons. Among 13 strains isolated, four were selected for their efficiency. Each of these four strains demonstrated a strong ability to grow as a ...

  16. Biocontrol properties of indigenous Trichoderma isolates from North ...

    African Journals Online (AJOL)

    Trichoderma is one of the best known and well described biocontrol fungi for its lytic activity and antagonistic properties against phytopathogens. In the present study, a total of 114 isolates of Trichoderma were isolated from the soil collected from diverse climatic conditions of Manipur on selective medium. Out of the total ...

  17. Isolation, identification and screening of potential cellulase-free ...

    African Journals Online (AJOL)

    In order to isolate cellulase-free xylanase-producing fungi, screening and isolation of fungi was done using decaying wood, agricultural wastes and other lignocellulosic wastes as microbial source. Thirty (30) fungal species were selected for further analysis based upon clearing zones formation on xylan enriched agar ...

  18. Isolation, purification and properties of lipase from Pseudomonas ...

    African Journals Online (AJOL)

    Six isolates (Ps1, Ps2, Ps3, Ps4, Ps5 and Ps6) producing lipase were screened from wastewater on a selective medium agar containing Tween 80 or olive oil as the only source of carbon. Isolate Ps5 showed the highest lipase activity which was later identified as Pseudomonas aeruginosa. The effect of media composition ...

  19. Enrichment and isolation of microbial strains degrading bioplastic ...

    African Journals Online (AJOL)

    acer

    2015-07-08

    Jul 8, 2015 ... Polyvinyl alcohol (PVA) degrading bacterial strains were isolated from various environmental sites rich in plastic wastes by using the enrichment culture technique. Among the various isolated strains, the selected potent PVA degrading bacterial strains were tentatively characterized as Bacillus and.

  20. Myopic Selection

    OpenAIRE

    Geroski, P. A.; Mazzucato, M.

    2002-01-01

    The severity of selection mechanisms and the myopia of selection are explored through a duopoly model where one firm tries to move down a learning curve in which costs are initially higher than its rival's but ultimately much lower. A trade-off is found between catch-up time and asymptotic market share: the more severe are selection pressures, the less likely is it that the learning technology will survive; however, if it does survive, the learning technology will in the limit be more competi...

  1. Myopic selection

    OpenAIRE

    Geroski, P.A.; Mazzucato, M.

    2002-01-01

    The severity of selection mechanisms and the myopia of selection are explored through a duopoly model where one firm tries to move down a learning curve in which costs are initially higher than its rival's but ultimately much lower. A trade-off is found between catch-up time and asymptotic market share: the more severe are selection pressures, the less likely is it that the learning technology will survive; however, if it does survive, the learning technology will in the limit be more competi...

  2. Selective mutism

    Science.gov (United States)

    ... have a family history of selective mutism, extreme shyness, or anxiety disorders, which may increase their risk ... well Inability to speak in certain social situations Shyness This pattern must be seen for at least ...

  3. Selective Enumeration

    National Research Council Canada - National Science Library

    Damon, Craig

    2000-01-01

    Selective enumeration is an approach to pruning search trees with the goal of preventing the generation of extraneous paths in the search tree, rather than generating paths that will later be pruned...

  4. Efficacy of Sakacin on Selected Food Pathogenic Microorganisms ...

    African Journals Online (AJOL)

    The efficacy of sakacin on selected food pathogenic microorganisms isolated from fermented milk products was investigated. The L .sake was isolated using the pour plate technique and was characterized based on it colony, cell morphology and some biochemical tests. This isolate was identified using standard scheme.

  5. Comparison of different selective enrichment steps to isolate Salmonella sp. from feces of finishing swine Comparação de diferentes etapas de enriquecimento seletivo no isolamento de Salmonella sp. de fezes de suínos de terminação

    Directory of Open Access Journals (Sweden)

    Geovana Brenner Michael

    2003-06-01

    Full Text Available A two-phase study was conducted to compare the efficacy of several enrichment selective-broth steps associated to different plating media for recovery of Salmonella sp. from finishing swine feces. In a first phase, Rappaport-Vassiliadis broth (RV incubated at 42ºC, Tetrathionate Müller-Kauffmann broth at 37ºC (TMK37 and 42ºC (TMK42, and Selenite Cystine broth (SC at 37ºC, in combination with three selective plating media Rambach agar (RA, Xylose-Lysine-Tergitol 4 agar (XLT4, and Brilliant-Green Phenol-Red Lactose Sucrose agar (VB were compared for recovery of Salmonella from artificially contaminated swine feces. In a second phase, RV, TMK37, and TMK42, associated with XLT4 and VB , were tested with naturally contaminated swine feces. In this study RV, TMK42 and TMK37 were superior to SC for isolating Salmonella sp. from artificially contaminated feces. TMK42 and RV were more productive than TMK37 for recovery of Salmonella from naturally contaminated feces samples. Selectivity and indication capability of the plating media were remarkably affected by the selective enrichment step effectiveness. The TMK42/XLT4 association was the most sensitive and RV/XLT4 the most specific. The use of VB agar is also recommended to increase the likelihood of isolating atypical H2S-late producing/ non-producing Salmonella. In this study RV and TMK42 were the most efficient selective enrichment for recovery of Salmonella sp. from swine feces.Através de um estudo em duas fases comparou-se a eficiência de etapas de enriquecimento seletivo, associadas a diferentes meios seletivos, na recuperação de Salmonella sp. de fezes de suínos de terminação. Em uma primeira fase, as amostras foram contaminadas artificialmente e os caldos Rappaport-Vassiliadis (RV incubado a 42ºC, Tetrationato Müller-Kauffmann a 37ºC (TMK37 e 42ºC (TMK42, e Selenito Cistina (SC a 37ºC foram testados, em associação com meios sólidos seletivos: ágar Rambach (RA, ágar Xilose

  6. Base isolation: Fresh insight

    Energy Technology Data Exchange (ETDEWEB)

    Shustov, V.

    1993-07-15

    The objective of the research is a further development of the engineering concept of seismic isolation. Neglecting the transient stage of seismic loading results in a widespread misjudgement: The force of resistance associated with velocity is mostly conceived as a source of damping vibrations, though it is an active force at the same time, during an earthquake type excitation. For very pliant systems such as base isolated structures with relatively low bearing stiffness and with artificially added heavy damping mechanism, the so called `damping`` force may occur even the main pushing force at an earthquake. Thus, one of the two basic pillars of the common seismic isolation philosophy, namely, the doctrine of usefulness and necessity of a strong damping mechanism, is turning out to be a self-deception, sometimes even jeopardizing the safety of structures and discrediting the very idea of seismic isolation. There is a way out: breaking with damping dependancy.

  7. isolated rural hospitals

    African Journals Online (AJOL)

    We report the results of a qualitative study that evaluated the effect of an educational intervention aimed at rural ... performance spiral in academically isolated rural hospitals. .... Heavy after-hrs workload, alcohol ... Continued academic stimula-.

  8. Alternativ isolering i bygninger

    DEFF Research Database (Denmark)

    Hansen, Ernst Jan de Place

    2001-01-01

    Resume af rapport om målinger på alternative isoleringsmaterialer i bygninger, udgivet af Statens Byggeforskningsinstitut (SBI meddelelse 128) under Energistyrelsens udviklingsprogram "Miljø- og arbejdsmiljøvenlig isolering"...

  9. Isolated Neutron Stars

    Directory of Open Access Journals (Sweden)

    Popov S.

    2010-10-01

    Full Text Available Several aspects related to astrophysics of isolated neutron stars are discussed. We start with an introduction into the “new zoo” of young isolated neutron stars. In addition to classical radio pulsars, now we know several species (soft gamma-ray repeators, anomalous X-ray pulsars, central compact objects in supernova remnants, close-by cooling neutron stars - aka “Magnificent seven”, - RRATs, and some others. All these types are briefly discussed. In the second lecture a description of magneto-rotational evolution of neutron stars is given. Finally, in the third lecture we discuss population synthesis of isolated neutron stars. In some details we discuss population synthesis of young isolated radio pulsars and young close-by cooling neutron stars.

  10. Isolation of Plastid Ribosomes.

    Science.gov (United States)

    Yamaguchi, Kenichi

    2017-01-01

    Plastid ribosomes are responsible for a large part of the protein synthesis in plant leaves, green algal cells, and the vast majority in the thalli of red algae. Plastid translation is necessary not only for photosynthesis but also for development/differentiation of plants and algae. While some isolated plastid ribosomes from a few green lineages have been characterized by biochemical and proteomic approaches, in-depth proteomics including analyses of posttranslational modifications and processing, comparative proteomics of plastid ribosomes isolated from the cells grown under different conditions, and those from different taxa are still to be carried out. Establishment of isolation methods for pure plastid ribosomes from a wider range of species would be beneficial to study the relationship between structure, function, and evolution of plastid ribosomes. Here I describe methodologies and provide example protocols for extraction and isolation of plastid ribosomes from a unicellular green alga (Chlamydomonas reinhardtii), a land plant (Arabidopsis thaliana), and a marine red macroalga (Pyropia yezoensis).

  11. Reproductive isolation during domestication.

    Science.gov (United States)

    Dempewolf, Hannes; Hodgins, Kathryn A; Rummell, Sonja E; Ellstrand, Norman C; Rieseberg, Loren H

    2012-07-01

    It has been hypothesized that reproductive isolation should facilitate evolution under domestication. However, a systematic comparison of reproductive barrier strength between crops and their progenitors has not been conducted to test this hypothesis. Here, we present a systematic survey of reproductive barriers between 32 economically important crop species and their progenitors to better understand the role of reproductive isolation during the domestication process. We took a conservative approach, avoiding those types of reproductive isolation that are poorly known for these taxa (e.g., differences in flowering time). We show that the majority of crops surveyed are isolated from their progenitors by one or more reproductive barriers, despite the fact that the most important reproductive barrier in natural systems, geographical isolation, was absent, at least in the initial stages of domestication for most species. Thus, barriers to reproduction between crops and wild relatives are closely associated with domestication and may facilitate it, thereby raising the question whether reproductive isolation could be viewed as a long-overlooked "domestication trait." Some of the reproductive barriers observed (e.g., polyploidy and uniparental reproduction), however, may have been favored for reasons other than, or in addition to, their effects on gene flow.

  12. The ISOL-MAFIOS Source (invited)

    Science.gov (United States)

    Geller, R.; Tamburella, C.; Belmont, J. L.

    1996-03-01

    ISOL-MAFIOS is a high-efficiency Min B ECRIS charge state breeder for short-lived ISOL beams in the PIAFE project. The ISOL species issuing from a bombarded target are locally ionized (1+) in a given ion source (ISOLDE type). They are then accelerated, magnetically selected, and transported in a long beam line, before being adequately introduced into ISOL-MAFIOS operating in continuous mode. In this system, an ECR plasma (created with a support gas) replaces the usual solid catcher. The ISOL-MAFIOS system captures the (1+) ions and transforms them into (q+) ions. If we call, overall efficiency of ISOL-MAFIOS the ratio of the beam intensities: I(q+) after the magnetic selector/I(1+) before entering in the source, we find for example for Rubidium IRb9+/IRb1+≊120 enA/1000 enA and a ratio at least two times larger for IKr9+/IKr1+. Moreover, an even more important result of ISOL-MAFIOS is that the release time+ionization time is of the order of 10-2 s instead of many seconds for a solid-state catcher. Thus in the PIAFE project isotopes whose half life times are ≳10-2 s become utilizable for acceleration in the two-cyclotron SARA. Note that in the PIAFE project the (1+) ISOL ions are produced by neutron bombardment of a U target inside the high flux reactor ILL and not by the bombardment of a target by a primary ion beam.

  13. Sampling bias created by ampicillin in isolation media for Aeromonas.

    Science.gov (United States)

    Huddleston, Jennifer R; Zak, John C; Jeter, Randall M

    2007-01-01

    Members of the bacterial genus Aeromonas are widely isolated from aquatic environments and studied in part for their ability to act as opportunistic pathogens in a variety of animals. All aeromonads, with the exception of Aeromonas trota, are generally thought to be resistant to ampicillin, so the antibiotic is frequently added to isolation medium as a selective agent. In this study, 282 aeromonads from environmental sources were isolated on a medium without ampicillin and their resistance to ampicillin determined. Of the 104 of these isolates that were judged to be independent (nonredundant), 18 (17.3%) were susceptible to ampicillin. A chi-square analysis was performed to determine the impact of ampicillin use on enumerating Aeromonas species from environmental samples. Our results indicate that, when ampicillin is used as a selective agent, a significant portion of the aeromonad population in at least some environments can be omitted from isolation.

  14. Isolation and selection of Bradyrhizobium from the root nodules of ...

    African Journals Online (AJOL)

    Makmal Sains Rumpai

    2012-07-19

    Jul 19, 2012 ... biofertilizer for providing greater stability in production .... Nodulation test by Leonard Bottle Jar Assembly (LBJA) was conducted in the Soil ... seedlings were placed in a tray filled with water and the roots were examined for ...

  15. Selective isolation and screening of fungi with herbicidal potential ...

    African Journals Online (AJOL)

    In irrigated agriculture, weed control through chemical herbicides, creates various environmental and health hazards which lead to the search for an alternate method of weed management, which is an eco-friendly and effective means. Biological control of weeds using microorganism is now extensively studied to control ...

  16. Fungi isolated in school buildings

    Directory of Open Access Journals (Sweden)

    Elżbieta Ejdys

    2013-12-01

    Full Text Available The aim of the study was to determine the species composition of fungi occurring on wall surfaces and in the air in school buildings. Fungi isolated from the air using the sedimentation method and from the walls using the surface swab technique constituted the study material. Types of finish materials on wall surfaces were identified and used in the analysis. Samples were collected in selected areas in two schools: classrooms, corridors, men's toilets and women's toilets, cloakrooms, sports changing rooms and shower. Examinations were conducted in May 2005 after the heating season was over. Fungi were incubated on Czapek-Dox medium at three parallel temperatures: 25, 37 and 40°C, for at least three weeks. A total of 379 isolates of fungi belonging to 32 genera of moulds, yeasts and yeast-like fungi were obtained from 321 samples in the school environment. The following genera were isolated most frequently: Aspergillus, Penicillium and Cladosporium. Of the 72 determined species, Cladosporium herbarum, Aspergillus fumigatus and Penicillium chrysogenum occurred most frequently in the school buildings. Wall surfaces were characterised by an increased prevalence of mycobiota in comparison with the air in the buildings, with a slightly greater species diversity. A certain species specificity for rough and smooth wall surfaces was demonstrated. Fungi of the genera Cladosporium and Emericella with large spores adhered better to smooth surfaces while those of the genus Aspergillus with smaller conidia adhered better to rough surfaces. The application of three incubation temperatures helped provide a fuller picture of the mycobiota in the school environment.

  17. Selective Competition

    NARCIS (Netherlands)

    A. Dubovik (Andrei); A. Parakhonyak (Alexei)

    2009-01-01

    textabstractWe consider a dynamic (differential) game with three players competing against each other. Each period each player can allocate his resources so as to direct his competition towards particular rivals -- we call such competition selective. The setting can be applied to a wide variety of

  18. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Maria Hernandez-Valladares

    2016-08-01

    Full Text Available Global mass spectrometry (MS-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC or metal oxide affinity chromatography (MOAC. We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

  19. Influenza virus isolation.

    Science.gov (United States)

    Krauss, Scott; Walker, David; Webster, Robert G

    2012-01-01

    The isolation of influenza viruses is important for the diagnosis of respiratory diseases in lower animals and humans, for the detection of the infecting agent in surveillance programs, and is an essential element in the development and production of vaccine. Since influenza is caused by a zoonotic virus it is necessary to do surveillance in the reservoir species (aquatic waterfowls), intermediate hosts (quails, pigs), and in affected mammals including humans. Two of the hemagglutinin (HA) subtypes of influenza A viruses (H5 and H7) can evolve into highly pathogenic (HP) strains for gallinaceous poultry; some HP H5 and H7 strains cause lethal infection of humans. In waterfowls, low pathogenic avian influenza (LPAI) isolates are obtained primarily from the cloaca (or feces); in domestic poultry, the virus is more often recovered from the respiratory tract than from cloacal samples; in mammals, the virus is most often isolated from the respiratory tract, and in cases of high pathogenic avian influenza (HPAI) from the blood and internal organs of infected birds. Virus isolation procedures are performed by inoculation of clinical specimens into embryonated eggs (primarily chicken eggs) or onto a variety of primary or continuous tissue culture systems. Successful isolation of influenza virus depends on the quality of the sample and matching the appropriate culture method to the sample type.

  20. Presença de substâncias fisològicamente ativas em culturas de E. Coli isolados das fezes de pacientes alérgicos pela técnica de Heist-Cohen Presence of substances with physiological activity in cultures of E. Coli isolated from stools in allergic patients by the Heist-Cohen pathogen selective method

    Directory of Open Access Journals (Sweden)

    João Canali Corrêa F.º

    1955-12-01

    Full Text Available Four patients with several allergic symptoms have been studied. Heist-Cohen pathogen selective method was positive in feces of all these patients for E. coli. Other