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Sample records for biosynthetic methods isolation

  1. Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

    Directory of Open Access Journals (Sweden)

    Zuiter Afnan

    2012-08-01

    Full Text Available Abstract Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

  2. The preparation of nucleotides uniformly labelled with carbon-14 by biosynthetic methods. Isolation of adenylic, uridylic, cytidylic,and guanylic acids, from the alkaline hydrolysate of escherichia coli RNA

    International Nuclear Information System (INIS)

    Garcia Pineda, M. D.; Pacheco Lopez, J.

    1978-01-01

    A method is described for the preparation and analysis of adenylic, uri dilic, cytidi- 11c and guanylic acids, labelled with 14 C . Escherichia coli cells have been labelled by growing them in a medi dia containing glucose-14 C as their only source of carbon. RNA is isolated from the cells, and after hydrolysis of the molecule the resulting nucleotides are separated by gel filtration and exchange chromatography. Chemical and radiochemical purity of the Isolated nucleotides is determined, and also its specific radioactivity. (Author) 30 refs

  3. Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium Haliangium ochraceum

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    Yuwei Sun

    2016-01-01

    Full Text Available Myxobacteria of marine origin are rare and hard-to-culture microorganisms, but they genetically harbor high potential to produce novel antibiotics. An extensive investigation on the secondary metabolome of the unique marine myxobacterium Haliangium ochraceum SMP-2 led to the isolation of a new polyketide-nonribosomal peptide hybrid product, haliamide (1. Its structure was elucidated by spectroscopic analyses including NMR and HR-MS. Haliamide (1 showed cytotoxicity against HeLa-S3 cells with IC50 of 12 μM. Feeding experiments were performed to identify the biosynthetic building blocks of 1, revealing one benzoate, one alanine, two propionates, one acetate and one acetate-derived terminal methylene. The biosynthetic gene cluster of haliamide (hla, 21.7 kbp was characterized through the genome mining of the producer, allowing us to establish a model for the haliamide biosynthesis. The sulfotransferase (ST-thioesterase (TE domains encoded in hlaB appears to be responsible for the terminal alkene formation via decarboxylation.

  4. An Improved in Vivo Deuterium Labeling Method for Measuring the Biosynthetic Rate of Cytokinins

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    Petr Tarkowski

    2010-12-01

    Full Text Available An improved method for determining the relative biosynthetic rate of isoprenoid cytokinins has been developed. A set of 11 relevant isoprenoid cytokinins, including zeatin isomers, was separated by ultra performance liquid chromatography in less than 6 min. The iP-type cytokinins were observed to give rise to a previously-unknown fragment at m/z 69; we suggest that the diagnostic (204-69 transition can be used to monitor the biosynthetic rate of isopentenyladenine. Furthermore, we found that by treating the cytokinin nucleotides with alkaline phosphatase prior to analysis, the sensitivity of the detection process could be increased. In addition, derivatization (propionylation improved the ESI-MS response by increasing the analytes' hydrophobicity. Indeed, the ESI-MS response of propionylated isopentenyladenosine was about 34% higher than that of its underivatized counterpart. Moreover, the response of the derivatized zeatin ribosides was about 75% higher than that of underivatized zeatin ribosides. Finally, we created a web-based calculator (IZOTOP that facilitates MS/MS data processing and offer it freely to the research community.

  5. Biosynthetic incorporation of [75Se]selenomethionine: a new method for labelling lymphocyte membrane antigens

    International Nuclear Information System (INIS)

    Dosseto, M.; Rohner, C.; Pierres, M.; Goridis, C.

    1981-01-01

    A novel approach for radiolabelling lymphocyte membrane antigens is described. This technique is based on the use of the γ-emitting amino acid analogue [ 75 Se]selenomethionine. Human HLA-A, B, C and DR heavy and light chains and mouse Ia antigens were efficiently labelled by this technique and were precipitated with monoclonal antibodies. Approximately the same radioactivity was incorporated into the HLA-A, B, C chains whether [ 75 Se]selenomethionine, [ 35 S]methionine or [ 3 H]leucine were used as precursors. Easily detectable as a γ-emitter, [ 75 Se]selenomethionine thus constitutes a useful biosynthetic label of lymphocyte surface antigens. The same method was used to label immunoglobulins produced by hybridomas and to determine the nature of the secreted light chains. (Auth.)

  6. A fast and simple GC MS method for lignan profiling in Anthriscus sylvestris and biosynthetically related plant species

    NARCIS (Netherlands)

    Koulman, A; Bos, R; Medarde, M; Pras, N; Quax, WJ

    2001-01-01

    A new GC-MS method for monitoring lignans was developed to study the variation in plants and elucidate the biosynthetic steps. A simple and fast extraction procedure for lyophilised plant material was developed, giving a lignan-rich extract. A GC-MS method was set up using an apolar WCOT fused

  7. Diverse and Abundant Secondary Metabolism Biosynthetic Gene Clusters in the Genomes of Marine Sponge Derived Streptomyces spp. Isolates

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    Stephen A. Jackson

    2018-02-01

    Full Text Available The genus Streptomyces produces secondary metabolic compounds that are rich in biological activity. Many of these compounds are genetically encoded by large secondary metabolism biosynthetic gene clusters (smBGCs such as polyketide synthases (PKS and non-ribosomal peptide synthetases (NRPS which are modular and can be highly repetitive. Due to the repeats, these gene clusters can be difficult to resolve using short read next generation datasets and are often quite poorly predicted using standard approaches. We have sequenced the genomes of 13 Streptomyces spp. strains isolated from shallow water and deep-sea sponges that display antimicrobial activities against a number of clinically relevant bacterial and yeast species. Draft genomes have been assembled and smBGCs have been identified using the antiSMASH (antibiotics and Secondary Metabolite Analysis Shell web platform. We have compared the smBGCs amongst strains in the search for novel sequences conferring the potential to produce novel bioactive secondary metabolites. The strains in this study recruit to four distinct clades within the genus Streptomyces. The marine strains host abundant smBGCs which encode polyketides, NRPS, siderophores, bacteriocins and lantipeptides. The deep-sea strains appear to be enriched with gene clusters encoding NRPS. Marine adaptations are evident in the sponge-derived strains which are enriched for genes involved in the biosynthesis and transport of compatible solutes and for heat-shock proteins. Streptomyces spp. from marine environments are a promising source of novel bioactive secondary metabolites as the abundance and diversity of smBGCs show high degrees of novelty. Sponge derived Streptomyces spp. isolates appear to display genomic adaptations to marine living when compared to terrestrial strains.

  8. Effect of immobilization stress on gene expression of catecholamine biosynthetic enzymes in heart auricles of socially isolated rats

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    L. Gavrilovic

    2009-12-01

    Full Text Available Chronic stress is associated with the development of cardiovascular diseases. The sympathoneural system plays an important role in the regulation of cardiac function both in health and disease. In the present study, the changes in gene expression of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH, dopamine-β-hydroxylase (DBH and phenylethanolamine N-methyltransferase (PNMT and protein levels in the right and left heart auricles of naive control and long-term (12 weeks socially isolated rats were investigated by Taqman RT-PCR and Western blot analysis. The response of these animals to additional immobilization stress (2 h was also examined. Long-term social isolation produced a decrease in TH mRNA level in left auricles (about 70% compared to the corresponding control. Expression of the DBH gene was markedly decreased both in the right (about 62% and left (about 81% auricles compared to the corresponding control, group-maintained rats, whereas PNMT mRNA levels remained unchanged. Exposure of group-housed rats to acute immobilization for 2 h led to a significant increase of mRNA levels of TH (about 267%, DBH (about 37% and PNMT (about 60% only in the right auricles. Additional 2-h immobilization of individually housed rats did not affect gene expression of these enzymes in either the right or left auricle. Protein levels of TH, DBH and PNMT in left and right heart auricles were unchanged either in both individually housed and immobilized rats. The unchanged mRNA levels of the enzymes examined after short-term immobilization suggest that the catecholaminergic system of the heart auricles of animals previously exposed to chronic psychosocial stress was adapted to maintain appropriate cardiovascular homeostasis.

  9. Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins

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    Antonia Susca

    2016-09-01

    Full Text Available The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB and ochratoxin A (OTA mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that i isolates of both species differed in ability to produce the mycotoxins; ii FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum cluster; iii FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and iv OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.

  10. An improved in vivo deuterium labeling method for measuring the biosynthetic rate of cytokinins

    Czech Academy of Sciences Publication Activity Database

    Tarkowski, Petr; Floková, K.; Václavíková, Kateřina; Jaworek, P.; Raus, M.; Nordström, A.; Novák, Ondřej; Doležal, Karel; Šebela, M.; Frébortová, Jitka

    2010-01-01

    Roč. 15, č. 12 (2010), s. 9214-9229 ISSN 1420-3049 R&D Projects: GA ČR(CZ) GA522/08/0920; GA MŠk ED0017/01/01; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : cytokinin * deuterium labelling * biosynthetic rate Subject RIV: CE - Biochemistry Impact factor: 1.988, year: 2010

  11. The preparation of nucleotides uniformly labelled with carbon-14 by biosynthetic methods. Isolation of adenylic, uridylic, cytidylic,and guanylic acids, from the alkaline hydrolysate of escherichia coli RNA; Preparacion de nucleiotidos uniformemente marcados con 14{sup C}, por via biosintetica. Aislamiento de los acidos adenilico, uridilico, citidilico y guanilico, procedentes de la hidrolisis alcalina de RNA de escherichia Coli.

    Energy Technology Data Exchange (ETDEWEB)

    Garcia Pineda, M D; Pacheco Lopez, J

    1978-07-01

    A method is described for the preparation and analysis of adenylic, uri dilic, cytidi- 11c and guanylic acids, labelled with 14{sup C}. Escherichia coli cells have been labelled by growing them in a medi dia containing glucose-14{sup C} as their only source of carbon. RNA is isolated from the cells, and after hydrolysis of the molecule the resulting nucleotides are separated by gel filtration and exchange chromatography. Chemical and radiochemical purity of the Isolated nucleotides is determined, and also its specific radioactivity. (Author) 30 refs.

  12. Schizosaccharomyces isolation method

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    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.

  13. Lactic Acid Bacterial Starter Culture with Antioxidant and γ-Aminobutyric Acid Biosynthetic Activities Isolated from Flatfish-Sikhae Fermentation.

    Science.gov (United States)

    Won, Yeong Geol; Yu, Hyun-Hee; Chang, Young-Hyo; Hwang, Han-Joon

    2015-12-01

    The aim of this study is to select a lactic acid bacterial strain as a starter culture for flatfish-Sikhae fermentation and to evaluate its suitability for application in a food system. Four strains of lactic acid bacteria isolated from commercial flatfish-Sikhae were identified and selected as starter culture candidates through investigation of growth rates, salt tolerance, food safety, and functional properties such as antioxidative and antimicrobial activities. The fermentation properties of the starter candidates were also examined in food systems prepared with these strains (candidate batch) in comparison with a spontaneous fermentation process without starter culture (control batch) at 15°C. The results showed that the candidate YG331 batch had better fermentation properties such as viable cell count, pH, and acidity than the other experimental batches, including the control batch. The results are expressed according to selection criteria based on a preliminary sensory evaluation and physiochemical investigation. Also, only a small amount of histamine was detected with the candidate YG331 batch. The radical scavenging activity of the candidate batches was better compared with the control batch, and especially candidate YG331 batch showed the best radical scavenging activity. Also, we isolated another starter candidate (identified as Lactobacillus brevis PM03) with γ-aminobutyric acid (GABA)-producing activity from commercial flatfish-Sikhae products. The sensory scores of the candidate YG331 batch were better than those of the other experimental batches in terms of flavor, color, and overall acceptance. In this study, we established selection criteria for the lactic acid bacterial starter for the flatfish-Sikhae production and finally selected candidate YG331 as the most suitable starter.

  14. A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins

    International Nuclear Information System (INIS)

    Plevin, Michael J.; Hamelin, Olivier; Boisbouvier, Jérôme; Gans, Pierre

    2011-01-01

    A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-[ 13 C]glucose and subsaturating amounts of 2-[ 13 C]methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional 1 H- 13 C correlation spectra. Protein samples are conveniently prepared using the same media composition as the main uniformly-labeled sample and contain higher levels of isotope-enrichment than fractional labeling approaches. This new strategy thus represents an economically-attractive, robust alternative for obtaining isotopically-encoded stereospecific NMR assignments of prochiral methyl groups.

  15. Review of the ISOL Method

    CERN Document Server

    Lindroos, M

    2004-01-01

    The ISOL technique was invented in Copenhagen over 50 years ago and eventually migrated to CERN where a suitable proton drive beam was available at the Syncho-Cyclotron. The quick spread of the technique from CERN to many other laboratories has resulted in a large user community, which has assured the continued development of the method, physics in the front-line of fundamental research and the application of the method to many applied sciences. The technique is today established as one of the main techniques for on-line isotope production of high intensity and high quality beams. The thick targets used allows the production of unmatched high intensity radioactive beams. The fact that the ions are produced at rest makes it ideally suitable for low energy experiments and for post acceleration using well established accelerator techniques. The many different versions of the technique will be discussed and the many facilities spread all over the world will be reviewed. The major developments at the existing faci...

  16. Assessing Social Isolation: Pilot Testing Different Methods.

    Science.gov (United States)

    Taylor, Harry Owen; Herbers, Stephanie; Talisman, Samuel; Morrow-Howell, Nancy

    2016-04-01

    Social isolation is a significant public health problem among many older adults; however, most of the empirical knowledge about isolation derives from community-based samples. There has been less attention given to isolation in senior housing communities. The objectives of this pilot study were to test two methods to identify socially isolated residents in low-income senior housing and compare findings about the extent of isolation from these two methods. The first method, self-report by residents, included 47 out of 135 residents who completed in-person interviews. To determine self-report isolation, residents completed the Lubben Social Network Scale 6 (LSNS-6). The second method involved a staff member who reported the extent of isolation on all 135 residents via an online survey. Results indicated that 26% of residents who were interviewed were deemed socially isolated by the LSNS-6. Staff members rated 12% of residents as having some or a lot of isolation. In comparing the two methods, staff members rated 2% of interviewed residents as having a lot of isolation. The combination of self-report and staff report could be more informative than just self-report alone, particularly when participation rates are low. However, researchers should be aware of the potential discrepancy between these two methods.

  17. Methods to isolate extracellular vesicles for diagnosis

    Science.gov (United States)

    Kang, Hyejin; Kim, Jiyoon; Park, Jaesung

    2017-12-01

    Extracellular vesicles (EVs) are small membrane-bound bodies that are released into extracellular space by diverse cells, and are found in body fluids like blood, urine and saliva. EVs contain RNA, DNA and proteins, which can be biomarkers for diagnosis. EVs can be obtained by minimally-invasive biopsy, so they are useful in disease diagnosis. High yield and purity contribute to precise diagnosis of disease, but damaged EVs and impurities can cause confu sed results. However, EV isolation methods have different yields and purities. Furthermore, the isolation method that is most suitable to maximize EV recovery efficiency depends on the experimental conditions. This review focuses on merits and demerits of several types of EV isolation methods, and provides examples of how to diagnose disease by exploiting information obtained by analysis of EVs.

  18. A new rapid method for isolating nucleoli.

    Science.gov (United States)

    Li, Zhou Fang; Lam, Yun Wah

    2015-01-01

    The nucleolus was one of the first subcellular organelles to be isolated from the cell. The advent of modern proteomic techniques has resulted in the identification of thousands of proteins in this organelle, and live cell imaging technology has allowed the study of the dynamics of these proteins. However, the limitations of current nucleolar isolation methods hinder the further exploration of this structure. In particular, these methods require the use of a large number of cells and tedious procedures. In this chapter we describe a new and improved nucleolar isolation method for cultured adherent cells. In this method cells are snap-frozen before direct sonication and centrifugation onto a sucrose cushion. The nucleoli can be obtained within a time as short as 20 min, and the high yield allows the use of less starting material. As a result, this method can capture rapid biochemical changes in nucleoli by freezing the cells at a precise time, hence faithfully reflecting the protein composition of nucleoli at the specified time point. This protocol will be useful for proteomic studies of dynamic events in the nucleolus and for better understanding of the biology of mammalian cells.

  19. K. OXYTOCA BACTERIOPHAGES ISOLATION METHODS IMPROVEMENT

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    G. R. Sadrtdinova

    2017-01-01

    Full Text Available The article presents the results of a study related to increasing the efficiency of phage isolation of bacteria of the species K. oxytoca, by developing the optimal composition of the medium used in the work. In scientific research, in almost all methods associated with the isolation of bacteriophages, meat-peptone broth and meat-peptone agar are used as the nutrient basis. The peculiarities of growth and cultivation of microorganisms create certain difficulties for the isolation of phages active against bacteria of the species K. oxytoca. The selection of components and the creation of an environment that would ensure the optimal growth of both the bacterial culture and the reproduction of the virus makes it possible to facilitate the isolation of bacteriophages. The number of bacterial strains used in the work was 7. All strains of cultures were obtained from the Museum of the Department of Microbiology, Virology, Epizootology and Veterinary and Sanitary Expertise of the Federal State Budget Educational Institution of Higher Education “Ulyanovsk State Agrarian University named after P.A. Stolypin”. The studies included 2 main stages. The first stage consisted in isolation of bacteriophages by the method of isolation from the external environment by the method of Adelson L.I., Lyashenko E.A. The material for the studies were samples: soil, sewage sample, fecal samples (2. Only 4 samples. According to the chosen method, the sowing of the putative phagolysate was carried out on meat-peptone agar (1.5% and the agar for isolating bacteriophages (Aph (1.5%. A positive result was the presence on the environment of negative colonies, clearly visible on the matt background of deep growth of bacteria. A negative result is a continuous growth (“lawn” of bacterial culture. As a control, the culture of the microorganism studied was used for the media. In the course of the conducted studies for the first stage, 2 bacteriophages were isolated, active

  20. Variation in fumonisin and ochratoxin production associated with differences in biosynthetic gene content in Aspergillus niger and A. welwitschiae isolates from multiple crop and geographic origins

    Science.gov (United States)

    The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both specie...

  1. Multi-development-HPTLC method for quantitation of hyoscyamine, scopolamine and their biosynthetic precursors in selected solanaceae plants grown in natural conditions and as in vitro cultures.

    Science.gov (United States)

    Jaremicz, Zbigniew; Luczkiewicz, Maria; Kisiel, Mariusz; Zárate, Rafael; El Jaber-Vazdekis, Nabil; Migas, Piotr

    2014-01-01

    Hyoscyamine and scopolamine, anti-cholinergic agents widely used in medicine, are typically obtained from plants grown under natural conditions. Since field cultivation entails certain difficulties (changeable weather, pests, etc.), attempts have been made to develop a plant in vitro culture system as an alternative source for the production of these compounds. During experiments to locate the limiting steps in the biotechnological procedure, it is important to monitor not only the levels of the final products but also the changes in the concentration of their precursors. To develop a HPTLC method for the separation and quantitation of the main tropane alkaloids hyoscyamine and scopolamine, their respective direct precursors littorine and anisodamine, and cuscohygrine, a product of a parallel biosynthetic pathway that shares a common precursor (N-methyl-∆(1) -pyrrolium cation) with tropane alkaloids. Using alkaloid extracts from Atropa baetica hairy roots, different TLC chromatographic systems and developing procedures were investigated. Full separation of all compounds was obtained on HPTLC Si60 F254 plates preconditioned with mobile phase vapours (chloroform:methanol:acetone:25% ammonia ratios of 75:15:10:1.8, v/v/v/v). The chromatograms were developed twice (at distances of 4.0 and 3.0 cm) in a Camag twin trough chamber and visualised with Dragendorff's reagent. Densitometric detection (λ = 190 and 520 nm) was used for quantitative analyses of the different plant samples. This method can be recommended for quantitation of hyoscyamine, scopolamine, anisodamine, littorine and cuscohygrine in different plant material (field grown vs. in vitro cultures). Copyright © 2013 John Wiley & Sons, Ltd.

  2. Water feeding method upon reactor isolation

    International Nuclear Information System (INIS)

    Sasaki, Koichi; Takahara, Kuniaki; Hamamura, Kenji; Arakawa, Masahiro.

    1990-01-01

    The present invention concerns a method of feeding water upon reactor isolation in a plural loop type reactor having a plurality of reactor cooling systems. Water can be injected to a plurality of pools even if the pressure between the pools is not balanced and the water level in the reactor cooling system is optimally controlled. That is, water can be injected in accordance with the amount required for each of the pools by setting the opening of a turbine inlet steam control valve to somewhat higher than the cooling system pressure of the highest pressure loop. Water feeding devices upon reactor isolation were required by the same number as that for the reactor cooling systems. Whereas since pumps and turbines are used in common without worsening the water injection controllability to each of the loops according to the method of this invention and, accordingly, the cost performance can be improved. Further, since the opening degree of the turbine inlet steam control valve is controlled while making the difference pressure constant between the turbine inlet pressure and the pump exhaust pressure, the amount of the turbine exhausted steams can be reduced and, further, water injection controllability of the flow rate control valve in the injection line is improved. (I.S.)

  3. Development of analysis methods for seismically isolated nuclear structures

    International Nuclear Information System (INIS)

    Yoo, Bong; Lee, Jae-Han; Koo, Gyeng-Hoi

    2002-01-01

    KAERI's contributions to the project entitled Development of Analysis Methods for Seismically Isolated Nuclear Structures under IAEA CRP of the intercomparison of analysis methods for predicting the behaviour of seismically isolated nuclear structures during 1996-1999 in effort to develop the numerical analysis methods and to compare the analysis results with the benchmark test results of seismic isolation bearings and isolated nuclear structures provided by participating countries are briefly described. Certain progress in the analysis procedures for isolation bearings and isolated nuclear structures has been made throughout the IAEA CRPs and the analysis methods developed can be improved for future nuclear facility applications. (author)

  4. Biosynthetic elongation of isolated teichuronic acid polymers via glucosyl- and N-acetylmannosaminuronosyltransferases from solubilized cytoplasmic membrane fragments of Micrococcus luteus.

    Science.gov (United States)

    Hildebrandt, K M; Anderson, J S

    1990-01-01

    Cytoplasmic membrane fragments of Micrococcus luteus catalyze in vitro biosynthesis of teichuronic acid from uridine diphosphate D-glucose (UDP-glucose), uridine diphosphate N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), and uridine diphosphate N-acetyl-D-glucosamine. Membrane fragments solubilized with Thesit (dodecyl alcohol polyoxyethylene ether) can utilize UDP-glucose and UDP-ManNAcA to effect elongation of teichuronic acid isolated from native cell walls. When UDP-glucose is the only substrate supplied, the detergent-solubilized glucosyltransferase incorporates a single glucosyl residue onto each teichuronic acid acceptor. When both UDP-glucose and UDP-ManNAcA are supplied, the glucosyltransferase and the N-acetylmannosaminuronosyltransferase act cooperatively to elongate the teichuronic acid acceptor by multiple additions of the disaccharide repeat unit. As shown by polyacrylamide gel electrophoresis, low-molecular-weight fractions of teichuronic acid are converted to higher-molecular-weight polymers by the addition of as many as 17 disaccharide repeat units. Images PMID:2118507

  5. The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

    Science.gov (United States)

    Demissie, Zerihun A; Erland, Lauren A E; Rheault, Mark R; Mahmoud, Soheil S

    2013-03-01

    Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

  6. Biosynthetic origin of acetic acid using SNIF-NMR

    International Nuclear Information System (INIS)

    Boffo, Elisangela Fabiana; Ferreira, Antonio Gilberto

    2006-01-01

    The main purpose of this work is to describe the use of the technique Site-Specific Natural Isotopic Fractionation of hydrogen (SNIF-NMR), using 2 H and 1 H NMR spectroscopy, to investigate the biosynthetic origin of acetic acid in commercial samples of Brazilian vinegar. This method is based on the deuterium to hydrogen ratio at a specific position (methyl group) of acetic acid obtained by fermentation, through different biosynthetic mechanisms, which result in different isotopic ratios. We measured the isotopic ratio of vinegars obtained through C 3 , C 4 , and CAM biosynthetic mechanisms, blends of C 3 and C 4 (agrins) and synthetic acetic acid. (author)

  7. A Modified Method for Isolation of Rhein from Senna

    Science.gov (United States)

    Mehta, Namita; Laddha, K. S.

    2009-01-01

    A simple and efficient method for the isolation of rhein from Cassia angustifolia (senna) leaves is described in which the hydrolysis of the sennosides and extraction of the hydrolysis products (free anthraquinones) is carried out in one step. Further isolation of rhein is achieved from the anthraquinone mixture. This method reduces the number of steps required for isolation of rhein as compared to conventional methods. PMID:20336207

  8. Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway

    DEFF Research Database (Denmark)

    Liu, Qing; Manzano, David; Tanić, Nikola

    2014-01-01

    Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew tha...

  9. Vanillin biosynthetic pathways in plants.

    Science.gov (United States)

    Kundu, Anish

    2017-06-01

    The present review compiles the up-to-date knowledge on vanillin biosynthesis in plant systems to focus principally on the enzymatic reactions of in planta vanillin biosynthetic pathway and to find out its impact and prospect in future research in this field. Vanillin, a very popular flavouring compound, is widely used throughout the world. The principal natural resource of vanillin is the cured vanilla pods. Due to the high demand of vanillin as a flavouring agent, it is necessary to explore its biosynthetic enzymes and genes, so that improvement in its commercial production can be achieved through metabolic engineering. In spite of significant advancement in elucidating vanillin biosynthetic pathway in the last two decades, no conclusive demonstration had been reported yet for plant system. Several biosynthetic enzymes have been worked upon but divergences in published reports, particularly in characterizing the crucial biochemical steps of vanillin biosynthesis, such as side-chain shortening, methylation, and glucoside formation and have created a space for discussion. Recently, published reviews on vanillin biosynthesis have focused mainly on the biotechnological approaches and bioconversion in microbial systems. This review, however, aims to compile in brief the overall vanillin biosynthetic route and present a comparative as well as comprehensive description of enzymes involved in the pathway in Vanilla planifolia and other plants. Special emphasis has been given on the key enzymatic biochemical reactions that have been investigated extensively. Finally, the present standpoint and future prospects have been highlighted.

  10. Targeting trichothecene biosynthetic genes

    NARCIS (Netherlands)

    Wei, Songhong; Lee, van der Theo; Verstappen, Els; Gent, van Marga; Waalwijk, Cees

    2017-01-01

    Biosynthesis of trichothecenes requires the involvement of at least 15 genes, most of which have been targeted for PCR. Qualitative PCRs are used to assign chemotypes to individual isolates, e.g., the capacity to produce type A and/or type B trichothecenes. Many regions in the core cluster

  11. Emergent biosynthetic capacity in simple microbial communities.

    Directory of Open Access Journals (Sweden)

    Hsuan-Chao Chiu

    2014-07-01

    Full Text Available Microbes have an astonishing capacity to transform their environments. Yet, the metabolic capacity of a single species is limited and the vast majority of microorganisms form complex communities and join forces to exhibit capabilities far exceeding those achieved by any single species. Such enhanced metabolic capacities represent a promising route to many medical, environmental, and industrial applications and call for the development of a predictive, systems-level understanding of synergistic microbial capacity. Here we present a comprehensive computational framework, integrating high-quality metabolic models of multiple species, temporal dynamics, and flux variability analysis, to study the metabolic capacity and dynamics of simple two-species microbial ecosystems. We specifically focus on detecting emergent biosynthetic capacity--instances in which a community growing on some medium produces and secretes metabolites that are not secreted by any member species when growing in isolation on that same medium. Using this framework to model a large collection of two-species communities on multiple media, we demonstrate that emergent biosynthetic capacity is highly prevalent. We identify commonly observed emergent metabolites and metabolic reprogramming patterns, characterizing typical mechanisms of emergent capacity. We further find that emergent secretion tends to occur in two waves, the first as soon as the two organisms are introduced, and the second when the medium is depleted and nutrients become limited. Finally, aiming to identify global community determinants of emergent capacity, we find a marked association between the level of emergent biosynthetic capacity and the functional/phylogenetic distance between community members. Specifically, we demonstrate a "Goldilocks" principle, where high levels of emergent capacity are observed when the species comprising the community are functionally neither too close, nor too distant. Taken together

  12. Study on design method for seismically isolated FBR plants

    International Nuclear Information System (INIS)

    Hirata, Kazuta; Yabana, Shuichi; Ohtori, Yasuki; Ishida, Katsuhiko; Sawada, Yoshihiro; Shiojiri; Hiroo; Mazda, Taiji

    1998-01-01

    CRIEPI conducted 'Demonstration test on FBR seismic isolation system' from 1987 to 1996 under contract with Ministry of International Trade and Industry, Japan. In the demonstration test, base isolation technologies are prepared and demonstrated to apply to FBR and the design guidelines are proposed. In this report overall contents of the design guidelines entitled Design guidelines for seismically base isolated FBR plants' are included. The design guidelines, as a rule, are limited to apply to FBR plants where entire reactor building is isolated in the horizontal direction using laminated rubber bearings as isolators. The design guidelines and its concepts, however, will be useful for the development of similar guidelines for other isolation systems using different type of isolation methods and other nuclear facilities. The design guidelines consist of three parts and appendices. The first part is 'Policy for Safety Design of Base Isolated FBR Plants' specifying the principles and the requirements in the planning and the design for the safety of base isolated FBR plants. The second part is Policy for Seismic Design of Base Isolated FBR' describing the principles and the requirements in the seismic design and the evaluation of safety for base isolated FBR plants. The third part is 'Design Methods for Seismic Isolated FBR Plants' detailing the methods, procedures and parameters to be used in the design and the evaluation of safety fro base isolated FBR plants. In appendices examples of design procedures for base isolated reactor building and laminated rubber bearings as well as various test data on laminated rubber bearings, etc. are shown. (author)

  13. A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

    Directory of Open Access Journals (Sweden)

    Belshoff Alex C

    2011-05-01

    Full Text Available Abstract Background Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes. Results We utilized a stable isotope resolved metabolomics (SIRM approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits

  14. Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

    Science.gov (United States)

    Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

    2008-06-01

    Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

  15. Biosynthetic Pathways of Ergot Alkaloids

    Directory of Open Access Journals (Sweden)

    Nina Gerhards

    2014-12-01

    Full Text Available Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines. All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine. Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes.

  16. Some Orchid Species Fungi Isolated by Different Methods

    Directory of Open Access Journals (Sweden)

    Arzu ÇIĞ

    2014-03-01

    Full Text Available Due to their very small seeds that do not contain endosperm, many terrestrial orchid species require the presence of fungi in order to germinate and maintain their lives; and symbiotic culture studies are being carried out on this topic. For the purpose of determining the orchid species on which the fungus to be used as inoculants in the symbiotic culture will be effective, fungi isolated through several isolation methods are cultured with orchid species. In this study a total of four different isolation methods were applied as one on the tubers and rhizomes and three on the soil of eleven orchid species from the Anacamptis, Cephalanthera, Dactylorhiza and Orchis genera. Three different culture media were used in the methods. At the end of the study Alternaria, Aspergillus, Fusarium, Macrophomina, Rhizoctonia, Trichoderma and Verticillium fungi were isolated. In the study that was conducted with the aimed to isolate particularly Rhizoctania spp. fungi, the fungi was isolated from the tubers of Dactylorhiza umbrosa and Orchis palustris species and the soil of the Orchis simia species. Fusarium and Aspergillus species were isolated the most in all implemented methods and from all species.

  17. Towards a Biosynthetic UAV

    Science.gov (United States)

    Block, Eli; Byemerwa, Jovita; Dispenza, Ross; Doughty, Benjamin; Gillyard, KaNesha; Godbole, Poorwa; Gonzales-Wright, Jeanette; Hull, Ian; Kannappan, Jotthe; Levine, Alexander; hide

    2014-01-01

    We are currently working on a series of projects towards the construction of a fully biological unmanned aerial vehicle (UAV) for use in scientific and humanitarian missions. The prospect of a biologically-produced UAV presents numerous advantages over the current manufacturing paradigm. First, a foundational architecture built by cells allows for construction or repair in locations where it would be difficult to bring traditional tools of production. Second, a major limitation of current research with UAVs is the size and high power consumption of analytical instruments, which require bulky electrical components and large fuselages to support their weight. By moving these functions into cells with biosensing capabilities - for example, a series of cells engineered to report GFP, green fluorescent protein, when conditions exceed a certain threshold concentration of a compound of interest, enabling their detection post-flight - these problems of scale can be avoided. To this end, we are working to engineer cells to synthesize cellulose acetate as a novel bioplastic, characterize biological methods of waterproofing the material, and program this material's systemic biodegradation. In addition, we aim to use an "amberless" system to prevent horizontal gene transfer from live cells on the material to microorganisms in the flight environment.

  18. Bioengineering natural product biosynthetic pathways for therapeutic applications.

    Science.gov (United States)

    Wu, Ming-Cheng; Law, Brian; Wilkinson, Barrie; Micklefield, Jason

    2012-12-01

    With the advent of next-generation DNA sequencing technologies, the number of microbial genome sequences has increased dramatically, revealing a vast array of new biosynthetic gene clusters. Genomics data provide a tremendous opportunity to discover new natural products, and also to guide the bioengineering of new and existing natural product scaffolds for therapeutic applications. Notably, it is apparent that the vast majority of biosynthetic gene clusters are either silent or produce very low quantities of the corresponding natural products. It is imperative therefore to devise methods for activating unproductive biosynthetic pathways to provide the quantities of natural products needed for further development. Moreover, on the basis of our expanding mechanistic and structural knowledge of biosynthetic assembly-line enzymes, new strategies for re-programming biosynthetic pathways have emerged, resulting in focused libraries of modified products with potentially improved biological properties. In this review we will focus on the latest bioengineering approaches that have been utilised to optimise yields and increase the structural diversity of natural product scaffolds for future clinical applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. A simple method for human peripheral blood monocyte Isolation

    Directory of Open Access Journals (Sweden)

    Marcos C de Almeida

    2000-04-01

    Full Text Available We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

  20. Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

    Science.gov (United States)

    Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

    2017-05-31

    clusters of pikromycin biosynthetic gene clusters. The 60 kb pikromycin biosynthetic gene cluster was isolated in a single integration pSBAC vector. Introduction of the pikromycin biosynthetic gene cluster into the pikromycin non-producing strains resulted in higher pikromycin production. The utility of the pSBAC system as a precise cloning tool for large-sized biosynthetic gene clusters was verified through heterologous expression of the pikromycin biosynthetic gene cluster. Moreover, this pSBAC-driven heterologous expression strategy was confirmed to be an ideal approach for production of low and inconsistent natural products such as pikromycin in S. venezuelae, implying that this strategy could be employed for development of a custom overexpression scheme of natural product biosynthetic gene clusters in actinomycetes.

  1. Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

    Science.gov (United States)

    Cirillo, J D; Weisbrod, T R; Banerjee, A; Bloom, B R; Jacobs, W R

    1994-07-01

    The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.

  2. Simple and efficient methods for isolation and activity measurement ...

    African Journals Online (AJOL)

    Jane

    2011-06-29

    Jun 29, 2011 ... Key words: Hirudin, thrombin titration method, chromatography, purification. INTRODUCTION. Since recombinant ... Escherichia coli in 1986, intensive research had been .... mixed with 50 µl sample was incubated in 37°C water for 5 min, then 5 µl .... conclusion, the concise and efficient isolation line of the.

  3. Rapid and inexpensive method for isolating plasmid DNA

    International Nuclear Information System (INIS)

    Aljanabi, S. M.; Al-Awadi, S. J.; Al-Kazaz, A. A.; Baghdad Univ.

    1997-01-01

    A small-scale and economical method for isolating plasmid DNA from bacteria is described. The method provides DNA of suitable quality for most DNA manipulation techniques. This DNA can be used for restriction endonuclease digestion, southern blot hybridization, nick translation and end labeling of DNA probes, Polymerase Chain Reaction (PCR) -based techniques, transformation, DNA cycle-sequencing, and Chain-termination method for DNA sequencing. The entire procedure is adapted to 1.5 ml microfuge tubes and takes approximately 30 mins. The DNA isolated by this method has the same purity produced by CTAB and cesium chloride precipitation and purification procedures respectively. The two previous methods require many hours to obtain the final product and require the use of very expensive equipment as ultracentrifuge. This method is well suited for the isolation of plasmid DNA from a large number of bacterial samples and in a very short time and low cost in laboratories where chemicals, expensive equipment and finance are limited factors in conducting molecular research. (authors). 11refs. 11refs

  4. Comparison of Various Methods for Isolation of Nocardia from Soil

    Directory of Open Access Journals (Sweden)

    Masoumeh Rasouli-Nasab

    2017-02-01

    Full Text Available Background The genus Nocardia is Gram-positive, aerobic filamentous bacilli and saprophytic micro-organisms that can be isolated from freshwater, salt water, dust and decaying vegetation especially the soil. This study aimed to investigate the several media for to determine a suitable culture media with the ability to better for the isolation of Nocardia from soil. Methods In this study, 400 soil samples were collected from different areas from Iran. The soil samples were then cultured on the four culture media such as Humic acid vitamin B agar, Paraffin agar, Sabouraud dextrose agar supplemented whit cycloheximide and carbon-free broth containing paraffin rods and incubated at 35°C. All of culture media investigated every 3 days for a month. Colonies suspicious to Nocardia were stained with Gram-stain, acid-fast and partially acid-fast and evaluated for resistance to lysozyme. Results From 400 soil samples, the number of 62, 10, 28 and 19 strains of Nocardia were isolated by paraffin rods, Humic Acid Vitamin B agar, Paraffin agar and Sabouraud dextrose agar whit cycloheximide, respectively. Most Nocardia strains were isolated using paraffin bait technique. Conclusions Isolation of Nocardia spp. is enhanced by using the paraffin baiting technique that relies on the selective ability of this micro-organism to metabolize paraffin.

  5. Preservation methods for isolates of ascochyta blight fungi

    Directory of Open Access Journals (Sweden)

    Joanna Marcinkowska

    2014-08-01

    Full Text Available Isolates of ascochyta blight fungi, two of Ascochyta pisi, four of Mycosphaerella pinodes and four of Phoma pinodella were stored: A - on slants under mineral oil, B - on CN's medium agar disks, and as conidial suspension: C - in glycerine, D · in water. Viability and pathogenicity of recovered cultures after each consecutive year were assesed from 1991 to 1999. The compared parameters were first of all strongly influenced by the preservation method, but fungus species and number of years had a minor importance. The best for longer storage was method "A" because after 9 years the isolates were viable, highly pathogenic, and cultures recovered from them were clean. Thc method "C'' is good for short keeping (2-3 years, as conidia in vials need only small space and gave clean cultures.

  6. Extraction Methods for the Isolation of Isoflavonoids from Plant Material

    Directory of Open Access Journals (Sweden)

    Blicharski Tomasz

    2017-03-01

    Full Text Available The purpose of this review is to describe and compare selected traditional and modern extraction methods employed in the isolation of isoflavonoids from plants. Conventional methods such as maceration, percolation, or Soxhlet extraction are still frequently used in phytochemical analysis. Despite their flexibility, traditional extraction techniques have significant drawbacks, including the need for a significant investment of time, energy, and starting material, and a requirement for large amounts of potentially toxic solvents. Moreover, these techniques are difficult to automate, produce considerable amount of waste and pose a risk of degradation of thermolabile compounds. Modern extraction methods, such as: ultrasound-assisted extraction, microwave-assisted extraction, accelerated solvent extraction, supercritical fluid extraction, and negative pressure cavitation extraction, can be regarded as remedies for the aforementioned problems. This manuscript discusses the use of the most relevant extraction techniques in the process of isolation of isoflavonoids, secondary metabolites that have been found to have a plethora of biological and pharmacological activities.

  7. Resource Isolation Method for Program’S Performance on CMP

    Science.gov (United States)

    Guan, Ti; Liu, Chunxiu; Xu, Zheng; Li, Huicong; Ma, Qiang

    2017-10-01

    Data center and cloud computing are more popular, which make more benefits for customers and the providers. However, in data center or clusters, commonly there is more than one program running on one server, but programs may interference with each other. The interference may take a little effect, however, the interference may cause serious drop down of performance. In order to avoid the performance interference problem, the mechanism of isolate resource for different programs is a better choice. In this paper we propose a light cost resource isolation method to improve program’s performance. This method uses Cgroups to set the dedicated CPU and memory resource for a program, aiming at to guarantee the program’s performance. There are three engines to realize this method: Program Monitor Engine top program’s resource usage of CPU and memory and transfer the information to Resource Assignment Engine; Resource Assignment Engine calculates the size of CPU and memory resource should be applied for the program; Cgroups Control Engine divide resource by Linux tool Cgroups, and drag program in control group for execution. The experiment result show that making use of the resource isolation method proposed by our paper, program’s performance can be improved.

  8. Method for strontium isolation from high-mineralized water

    International Nuclear Information System (INIS)

    Evzhanov, Kh.; Andriyasova, G.M.

    1983-01-01

    A method to isolate strontium from high-mineralized waters containing sodium, magnesium, calcium and strontium chlorides, which differ from the prototype method in a considerable decrease in energy consumption with the preservation of a high degree of Sr, Mg and Ca isolation selectivity, has been suggested. According to the method suggested mineralized waters are treated with alkali (NaOH) in the amount of 95-97% of stoichiometry by magnesium, then after separation of magnesium hydroxide precipitate mother liquor is treated with sodium carbonate in the amount of 50-60% of stoichiometry by calcium. After separation of calcium carbonate precipitate mother liquor is treated with NaOH in the amount of 130-135% of stoichiometry by calcium. After separation of calcium hydroxide precipitate from mother liquor by means of sodium carbonate introduction strontium carbonate is isolated. The degree of strontium extraction in the form of SrCO 3 constitutes 90.5% of its content in the initial solution. The method presented can be used for strontium separation from natural and waste waters

  9. A modified efficient method for dental pulp stem cell isolation.

    Science.gov (United States)

    Raoof, Maryam; Yaghoobi, Mohammad Mehdi; Derakhshani, Ali; Kamal-Abadi, Ali Mohammadi; Ebrahimi, Behnam; Abbasnejad, Mehdi; Shokouhinejad, Noushin

    2014-03-01

    Dental pulp stem cells can be used in regenerative endodontic therapy. The aim of this study was to introduce an efficient method for dental pulp stem cells isolation. In this in-vitro study, 60 extracted human third molars were split and pulp tissue was extracted. Dental pulp stem cells were isolated by the following three different methods: (1) digestion of pulp by collagenase/dispase enzyme and culture of the released cells; (2) outgrowth of the cells by culture of undigested pulp pieces; (3) digestion of pulp tissue pieces and fixing them. The cells were cultured in minimum essential medium alpha modification (αMEM) medium supplemented with 20% fetal bovine serum(FBS) in humid 37°C incubator with 5% CO 2. The markers of stem cells were studied by reverse transcriptase polymerase chain reaction (PCR). The student t-test was used for comparing the means of independent groups. P third method, we obtained stem cells successfully with about 60% efficiency after 2 days. The results of RT-PCR suggested the expression of Nanog, Oct-4, and Nucleostemin markers in the isolated cells from dental pulps. This study proposes a new method with high efficacy to obtain dental pulp stem cells in a short time.

  10. A simple method for DNA isolation from Xanthomonas spp.

    Directory of Open Access Journals (Sweden)

    Gomes Luiz Humberto

    2000-01-01

    Full Text Available A simple DNA isolation method was developed with routine chemicals that yields high quality and integrity preparations when compared to some of the most well known protocols. The method described does not require the use of lysing enzymes, water bath and the DNA was obtained within 40 minutes The amount of nucleic acid extracted (measured in terms of absorbancy at 260 nm from strains of Xanthomonas spp., Pseudomonas spp. and Erwinia spp. was two to five times higher than that of the most commonly used method.

  11. An improved method for isolating viruses from asymptomatic carrier fish

    Science.gov (United States)

    Amend, Donald F.; Pietsch, John P.

    1972-01-01

    This paper describes a method using elevated levels of penicillin, streptomycin, and nystatin instead of filters to control bacteria and mold contaminants in specimens processed for virus isolation. Filters were shown to significantly reduce the virus concentration. Virus and tissue cultures were not affected by this procedure. In field tests nearly three times more specimens were positive for virus with this method than with the widely used filter technique. Moreover, the cost of materials was less. This method is recommended for inspection and certification purposes.

  12. A modified efficient method for dental pulp stem cell isolation

    Directory of Open Access Journals (Sweden)

    Maryam Raoof

    2014-01-01

    Full Text Available Background: Dental pulp stem cells can be used in regenerative endodontic therapy. The aim of this study was to introduce an efficient method for dental pulp stem cells isolation. Materials and Methods: In this in-vitro study, 60 extracted human third molars were split and pulp tissue was extracted. Dental pulp stem cells were isolated by the following three different methods: (1 digestion of pulp by collagenase/dispase enzyme and culture of the released cells; (2 outgrowth of the cells by culture of undigested pulp pieces; (3 digestion of pulp tissue pieces and fixing them. The cells were cultured in minimum essential medium alpha modification (αMEM medium supplemented with 20% fetal bovine serum(FBS in humid 37°C incubator with 5% CO 2 . The markers of stem cells were studied by reverse transcriptase polymerase chain reaction (PCR. The student t-test was used for comparing the means of independent groups. P <0.05 was considered as significant. Results: The results indicated that by the first method a few cell colonies with homogenous morphology were detectable after 4 days, while in the outgrowth method more time was needed (10-12 days to allow sufficient numbers of heterogeneous phenotype stem cells to migrate out of tissue. Interestingly, with the improved third method, we obtained stem cells successfully with about 60% efficiency after 2 days. The results of RT-PCR suggested the expression of Nanog, Oct-4, and Nucleostemin markers in the isolated cells from dental pulps. Conclusion: This study proposes a new method with high efficacy to obtain dental pulp stem cells in a short time.

  13. Accumulation of Rutin and Betulinic Acid and Expression of Phenylpropanoid and Triterpenoid Biosynthetic Genes in Mulberry (Morus alba L.).

    Science.gov (United States)

    Zhao, Shicheng; Park, Chang Ha; Li, Xiaohua; Kim, Yeon Bok; Yang, Jingli; Sung, Gyoo Byung; Park, Nam Il; Kim, Soonok; Park, Sang Un

    2015-09-30

    Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food source of the silkworm. Here, 21 cDNAs encoding phenylpropanoid biosynthetic genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from mulberry. The expression levels of genes involved in these biosynthetic pathways and the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites, were investigated in different plant organs. Most phenylpropanoid and triterpene biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were downregulated during fruit ripening. The accumulation of rutin was more than fivefold higher in leaves than in other organs, and higher levels of betulin and betulinic acid were found in roots and leaves than in fruit. By comparing the contents of these compounds with gene expression levels, we speculate that MaUGT78D1 and MaLUS play important regulatory roles in the rutin and betulin biosynthetic pathways.

  14. An optimized method for mouse liver sinusoidal endothelial cell isolation

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

    2016-12-10

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  15. An optimized method for mouse liver sinusoidal endothelial cell isolation

    International Nuclear Information System (INIS)

    Meyer, Jeremy; Lacotte, Stéphanie; Morel, Philippe; Gonelle-Gispert, Carmen; Bühler, Léo

    2016-01-01

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  16. Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

    DEFF Research Database (Denmark)

    Liu, Chengwei; Tagami, Koichi; Minami, Atsushi

    2015-01-01

    KULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products....

  17. Radical SAM, A Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods

    Energy Technology Data Exchange (ETDEWEB)

    Sofia, Heidi J.; Chen, Guang; Hetzler, Elizabeth G.; Reyes Spindola, Jorge F.; Miller, Nancy E.

    2001-03-01

    A large protein superfamily with over 500 members has been discovered and analyzed using powerful new bioinformatics and information visualization methods. Evidence exists that these proteins generate a 5?-deoxyadenosyl radical by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. Radical SAM superfamily proteins function in DNA precursor, vitamin, cofactor, antibiotic, and herbicide biosynthesis in a collection of basic and familiar pathways. One of the members is interferon-inducible and is considered a candidate drug target for osteoporosis. The identification of this superfamily suggests that radical-based catalysis is important in a number of previously well-studied but unresolved biochemical pathways.

  18. A molecular method for typing Herpes simplex virus isolates as an alternative to immunofluorescence methods

    Directory of Open Access Journals (Sweden)

    Abraham A

    2009-01-01

    Full Text Available Background: Typing of Herpes simplex virus (HSV isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF assays and polymerase chain reaction (PCR. This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb based IF test. Study design : This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase ( pol gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42 of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42 were HSV-2, and two (4.8% were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30, whereas the cost per MAb IF test is about Rs 1,500 (USD 35 including all overheads (reagents, instruments, personnel time, and consumables. Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers is now more widespread than fluorescence microscopes in a country like India.

  19. Targeting the GPI biosynthetic pathway.

    Science.gov (United States)

    Yadav, Usha; Khan, Mohd Ashraf

    2018-02-27

    The GPI (Glycosylphosphatidylinositol) biosynthetic pathway is a multistep conserved pathway in eukaryotes that culminates in the generation of GPI glycolipid which in turn anchors many proteins (GPI-APs) to the cell surface. In spite of the overall conservation of the pathway, there still exist subtle differences in the GPI pathway of mammals and other eukaryotes which holds a great promise so far as the development of drugs/inhibitors against specific targets in the GPI pathway of pathogens is concerned. Many of the GPI structures and their anchored proteins in pathogenic protozoans and fungi act as pathogenicity factors. Notable examples include GPI-anchored variant surface glycoprotein (VSG) in Trypanosoma brucei, GPI-anchored merozoite surface protein 1 (MSP1) and MSP2 in Plasmodium falciparum, protein-free GPI related molecules like lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) in Leishmania spp., GPI-anchored Gal/GalNAc lectin and proteophosphoglycans in Entamoeba histolytica or the GPI-anchored mannoproteins in pathogenic fungi like Candida albicans. Research in this active area has already yielded encouraging results in Trypanosoma brucei by the development of parasite-specific inhibitors of GlcNCONH 2 -β-PI, GlcNCONH 2 -(2-O-octyl)-PI and salicylic hydroxamic acid (SHAM) targeting trypanosomal GlcNAc-PI de-N-acetylase as well as the development of antifungal inhibitors like BIQ/E1210/gepinacin/G365/G884 and YW3548/M743/M720 targeting the GPI specific fungal inositol acyltransferase (Gwt1) and the phosphoethanolamine transferase-I (Mcd4), respectively. These confirm the fact that the GPI pathway continues to be the focus of researchers, given its implications for the betterment of human life.

  20. Deep sequencing as a method of typing bluetongue virus isolates.

    Science.gov (United States)

    Rao, Pavuluri Panduranga; Reddy, Yella Narasimha; Ganesh, Kapila; Nair, Shreeja G; Niranjan, Vidya; Hegde, Nagendra R

    2013-11-01

    Bluetongue (BT) is an economically important endemic disease of livestock in tropics and subtropics. In addition, its recent spread to temperate regions like North America and Northern Europe is of serious concern. Rapid serotyping and characterization of BT virus (BTV) is an essential step in the identification of origin of the virus and for controlling the disease. Serotyping of BTV is typically performed by serum neutralization, and of late by nucleotide sequencing. This report describes the near complete genome sequencing and typing of two isolates of BTV using Illumina next generation sequencing platform. Two of the BTV RNAs were multiplexed with ten other unknown samples. Viral RNA was isolated and fragmented, reverse transcribed, the cDNA ends were repaired and ligated with a multiplex oligo. The genome library was amplified using primers complementary to the ligated oligo and subjected to single and paired end sequencing. The raw reads were assembled using a de novo method and reference-based assembly was performed based on the contig data. Near complete sequences of all segments of BTV were obtained with more than 20× coverage, and single read sequencing method was sufficient to identify the genotype and serotype of the virus. The two viruses used in this study were typed as BTV-1 and BTV-9E. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Characterization of the fumonisin B2 biosynthetic gene cluster in Aspergillus niger and A. awamori.

    Science.gov (United States)

    Aspergillus niger and A. awamori strains isolated from grapes cultivated in Mediterranean basin were examined for fumonisin B2 (FB2) production and presence/absence of sequences within the fumonisin biosynthetic gene (fum) cluster. Presence of 13 regions in the fum cluster was evaluated by PCR assay...

  2. Elucidation of the biosynthetic pathway for the production of the pigment chrysogine by Penicillium chrysogenum

    NARCIS (Netherlands)

    Viggiano, Annarita; Salo, Oleksandr; Ali, Hazrat; Szymanski, Wiktor; Lankhorst, Peter P; Nygård, Yvonne; Bovenberg, Roel A L; Driessen, Arnold J M

    Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although it was first isolated in 1973, the biosynthetic pathway has so far not been resolved. Here, we show that the deletion of the highly expressed non-ribosomal peptide synthetase (NRPS) gene

  3. ADVANCED SEISMIC BASE ISOLATION METHODS FOR MODULAR REACTORS

    Energy Technology Data Exchange (ETDEWEB)

    E. Blanford; E. Keldrauk; M. Laufer; M. Mieler; J. Wei; B. Stojadinovic; P.F. Peterson

    2010-09-20

    Advanced technologies for structural design and construction have the potential for major impact not only on nuclear power plant construction time and cost, but also on the design process and on the safety, security and reliability of next generation of nuclear power plants. In future Generation IV (Gen IV) reactors, structural and seismic design should be much more closely integrated with the design of nuclear and industrial safety systems, physical security systems, and international safeguards systems. Overall reliability will be increased, through the use of replaceable and modular equipment, and through design to facilitate on-line monitoring, in-service inspection, maintenance, replacement, and decommissioning. Economics will also receive high design priority, through integrated engineering efforts to optimize building arrangements to minimize building heights and footprints. Finally, the licensing approach will be transformed by becoming increasingly performance based and technology neutral, using best-estimate simulation methods with uncertainty and margin quantification. In this context, two structural engineering technologies, seismic base isolation and modular steel-plate/concrete composite structural walls, are investigated. These technologies have major potential to (1) enable standardized reactor designs to be deployed across a wider range of sites, (2) reduce the impact of uncertainties related to site-specific seismic conditions, and (3) alleviate reactor equipment qualification requirements. For Gen IV reactors the potential for deliberate crashes of large aircraft must also be considered in design. This report concludes that base-isolated structures should be decoupled from the reactor external event exclusion system. As an example, a scoping analysis is performed for a rectangular, decoupled external event shell designed as a grillage. This report also reviews modular construction technology, particularly steel-plate/concrete construction using

  4. ADVANCED SEISMIC BASE ISOLATION METHODS FOR MODULAR REACTORS

    International Nuclear Information System (INIS)

    Blanford, E.; Keldrauk, E.; Laufer, M.; Mieler, M.; Wei, J.; Stojadinovic, B.; Peterson, P.F.

    2010-01-01

    Advanced technologies for structural design and construction have the potential for major impact not only on nuclear power plant construction time and cost, but also on the design process and on the safety, security and reliability of next generation of nuclear power plants. In future Generation IV (Gen IV) reactors, structural and seismic design should be much more closely integrated with the design of nuclear and industrial safety systems, physical security systems, and international safeguards systems. Overall reliability will be increased, through the use of replaceable and modular equipment, and through design to facilitate on-line monitoring, in-service inspection, maintenance, replacement, and decommissioning. Economics will also receive high design priority, through integrated engineering efforts to optimize building arrangements to minimize building heights and footprints. Finally, the licensing approach will be transformed by becoming increasingly performance based and technology neutral, using best-estimate simulation methods with uncertainty and margin quantification. In this context, two structural engineering technologies, seismic base isolation and modular steel-plate/concrete composite structural walls, are investigated. These technologies have major potential to (1) enable standardized reactor designs to be deployed across a wider range of sites, (2) reduce the impact of uncertainties related to site-specific seismic conditions, and (3) alleviate reactor equipment qualification requirements. For Gen IV reactors the potential for deliberate crashes of large aircraft must also be considered in design. This report concludes that base-isolated structures should be decoupled from the reactor external event exclusion system. As an example, a scoping analysis is performed for a rectangular, decoupled external event shell designed as a grillage. This report also reviews modular construction technology, particularly steel-plate/concrete construction using

  5. An improved method for isolation of RNA from bone

    Directory of Open Access Journals (Sweden)

    Carter Lauren E

    2012-01-01

    Full Text Available Abstract Background Bone physiology is increasingly appreciated as an important contributor to metabolic disorders such as type 2 diabetes. However, progress in understanding the role of bone in determining metabolic health is hampered by the well-described difficulty of obtaining high quality RNA from bone for gene expression analysis using the currently available approaches. Results We developed a simple approach to isolate bone RNA that combines pulverizing the bone and the phenol-guanidinium based RNA extraction in a single step while maintaining near-freezing temperatures. This single step method increases the yield of high quality RNA by eight-fold, with RNA integrity numbers ranging from 6.7 to 9.2. Conclusions Our streamlined approach substantially increases the yield of high-quality RNA from bone tissue while facilitating safe and efficient processing of multiple samples using readily available platforms. The RNA obtained from this method is suitable for use in gene expression analysis in real-time quantitative PCR, microarray, and next generation sequencing applications.

  6. Total error components - isolation of laboratory variation from method performance

    International Nuclear Information System (INIS)

    Bottrell, D.; Bleyler, R.; Fisk, J.; Hiatt, M.

    1992-01-01

    The consideration of total error across sampling and analytical components of environmental measurements is relatively recent. The U.S. Environmental Protection Agency (EPA), through the Contract Laboratory Program (CLP), provides complete analyses and documented reports on approximately 70,000 samples per year. The quality assurance (QA) functions of the CLP procedures provide an ideal data base-CLP Automated Results Data Base (CARD)-to evaluate program performance relative to quality control (QC) criteria and to evaluate the analysis of blind samples. Repetitive analyses of blind samples within each participating laboratory provide a mechanism to separate laboratory and method performance. Isolation of error sources is necessary to identify effective options to establish performance expectations, and to improve procedures. In addition, optimized method performance is necessary to identify significant effects that result from the selection among alternative procedures in the data collection process (e.g., sampling device, storage container, mode of sample transit, etc.). This information is necessary to evaluate data quality; to understand overall quality; and to provide appropriate, cost-effective information required to support a specific decision

  7. Structural Diversification of Lyngbyatoxin A by Host-Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster.

    Science.gov (United States)

    Zhang, Lihan; Hoshino, Shotaro; Awakawa, Takayoshi; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-08-03

    Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. On the biosynthetic origin of carminic acid

    DEFF Research Database (Denmark)

    Rasmussen, Silas A.; Kongstad, Kenneth T; Khorsand-Jamal, Paiman

    2018-01-01

    provides solid evidence of a polyketide, rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present a detailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all described coccid pigments which share a flavokermesic...... distribution suggests a common evolutionary origin for the trait in all coccid dye producing insect species....

  9. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.

    2013-01-23

    Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

  10. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.; Xü , Ying; Lu, Liang; Kersten, Roland D.; Shao, Zongze; Al-Suwailem, Abdulaziz M.; Dorrestein, Pieter C.; Qian, Peiyuan; Moore, Bradley S.

    2013-01-01

    Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus. © 2012 American Chemical Society.

  11. Radioactive ion beam production by the ISOL method for SPIRAL

    International Nuclear Information System (INIS)

    Landre-Pellemoine, Frederique

    2001-01-01

    This work is directly related to the SPIRAL project (Systeme de Production d'Ions Radioactifs Acceleres en Lignes) of which the start up will begin in September 2001 at GANIL (Grand Accelerateur National d'Ions Lourds) in Caen. This thesis primarily concerns the development of radioactive ion production systems (target/ion source) by the thorough study of each production stage of the ISOL (Isotopic Separation On Line) method: target and/or projectile fragmentation production, diffusion out of target material, effusion into the ion source and finally the ionization of the radioactive atoms. A bibliographical research and thermal simulations allowed us to optimize materials and the shape of the production and diffusion targets. A first target was optimized and made reliable for the radioactive noble gases production (argon, neon...). A second target dedicated to the radioactive helium production was entirely designed and realised (from the specifications to the 'off line' and 'on line' tests). Finally, a third target source system was defined for singly-charged radioactive alkaline production. The intensities of secondary beams planned for SPIRAL are presented here. A detailed study of the diffusion effusion efficiency for these various targets showed that the use of a fine microstructure carbon (grain size of 1 μm) improved the diffusion and showed the importance of thickness of the lamella for the short lived isotope effusion. (author) [fr

  12. Use of [75Se]selenomethionine in immunoglobulin biosynthetic studies

    International Nuclear Information System (INIS)

    Gutman, G.A.; Warner, N.L.; Harris, A.W.; Bowles, A.

    1978-01-01

    The gamma-emitting amino acid analog, [ 75 Se] selenomethionine, has been used as a biosynthetic label for immunoglobulins secreted by plasmacytomas in tissue culture. The secreted products are structurally intact with respect to their antibody combining sites and their class and allotype antigenic specificities. A component of [ 75 Se] selenomethionine preparations was found to bind to fetal calf serum proteins, in a manner releasable by mercaptoethanol, but not by sodium dodecyl sulfate and urea. Methods for circumventing the problems caused by this binding are described. (Auth.)

  13. A fast method for large-scale isolation of phages from hospital ...

    African Journals Online (AJOL)

    This plaque-forming method could be adopted to isolate E. coli phage easily, rapidly and in large quantities. Among the 18 isolated E. coli phages, 10 of them had a broad host range in E. coli and warrant further study. Key words: Escherichia coli phages, large-scale isolation, drug resistance, biological properties.

  14. [New isolation methods and phylogenetic diversity of actinobacteria from hypersaline beach in Aksu].

    Science.gov (United States)

    Zhang, Yao; Xia, Zhanfeng; Cao, Xinbo; Li, Jun; Zhang, Lili

    2013-08-04

    We explored 4 new methods to improve the isolation of actinobacterial resources from high salt areas. Optimized media based on 4 new strategies were used for isolating actinobacteria from hypersaline beaches. Glycerin-arginine, trehalose-creatine, glycerol-asparticacid, mannitol-casein, casein-mannitol, mannitol-alanine, chitosan-asparagineand GAUZE' No. 1 were used as basic media. New isolation strategy includes 4 methods: ten-fold dilution culture, simulation of the original environment, actinobacterial culture guided by uncultured molecular technology detected, and reference of actinobacterial media for brackish marine environment. The 16S rRNA genes of the isolates were amplified with bacterial universal primers. The results of 16S rRNA gene sequences were compared with sequences obtained from GenBank databases. We constructed phylogenetic tree with the neighbor-joining method. No actinobacterial strains were isolated by 8 media of control group, while 403 strains were isolated by new strategies. The isolates by new methods were members of 14 genera (Streptomyces, Streptomonospora, Saccharomonospora, Plantactinospora, Nocardia, Amycolatopsis, Glycomyces, Micromonospora, Nocardiopsis, Isoptericola, Nonomuraea, Thermobifida, Actinopolyspora, Actinomadura) of 10 families in 8 suborders. The most abundant and diverse isolates were the two suborders of Streptomycineae (69.96%) and Streptosporangineaesuborder (9.68%) within the phylum Actinobacteria, including 9 potential novel species. New isolation methods significantly improved the actinobacterial culturability of hypersaline areas, and obtained many potential novel species, which provided a new and more effective way to isolate actinobacteria resources in hypersaline environments.

  15. Development of an evaluation method for seismic isolation systems of nuclear power facilities. Development of crossover piping design method for seismic isolation systems

    International Nuclear Information System (INIS)

    Otoyo, Teruyoshi; Otani, Akihito; Otani, Akihito; Fukushima, Shunsuke; Jimbo, Masakazu; Yamamoto, Tomofumi; Sakakida, Takaaki; Onishi, Shigenobu

    2014-01-01

    In the conceptual design of seismic isolation systems of nuclear power facilities, there exist two types of installation. The first type is to isolate both the reactor and the turbine buildings, the other is to isolate only the reactor building. In the latter type, the crossover piping, which installed between the isolated and the non-isolated buildings, is excited and deformed by the different motions of those buildings. In this study, shaking tests of 1/10 scaled model of the main steam piping and FEM analyses under multiple support excitation conditions have been performed to investigate the vibration behavior of the crossover piping. It was confirmed that modal time-history analyses could be in good agreement with the shaking test results. Also, Numerous combination methods were investigated by comparing response spectrum analyses and modal time-history analyses. In conclusion, response spectrum analyses using SRSS combinations could correspond to time-history analyses. (author)

  16. Methods for extracellular vesicles isolation in a hospital setting

    Directory of Open Access Journals (Sweden)

    Matías eSáenz-Cuesta

    2015-02-01

    Full Text Available The research in extracellular vesicles (EVs has been rising during the last decade. However, there is no clear consensus on the most accurate protocol to isolate and analyze them. Besides, most of the current protocols are difficult to implement in a hospital setting due to being very time consuming or to requirements of specific infrastructure. Thus, our aim is to compare five different protocols (comprising two different medium-speed differential centrifugation protocols; commercially polymeric precipitation -exoquick-; acid precipitation; and ultracentrifugation for blood and urine samples to determine the most suitable one for the isolation of EVs. Nanoparticle tracking analysis, flow cytometry, western blot, electronic microscopy and spectrophotometry were used to characterize basic aspects of EVs such us concentration, size distribution, cell-origin and transmembrane markers and RNA concentration. The highest EV concentrations were obtained using the exoquick protocol, followed by both differential centrifugation protocols, while the ultracentrifugation and acid-precipitation protocols yielded considerably lower EV concentrations. The five protocols isolated EVs of similar characteristics regarding markers and RNA concentration however standard protocol recovered only small EVs. EV isolated with exoquick presented difficult to be analyzed with western blot. The RNA concentrations obtained from urine-derived EVs were similar to those obtained from blood-derived ones, despite the urine EV concentration being 10 to 20 times lower. We consider that a medium-speed differential centrifugation could be suitable to be applied in a hospital setting due to require the simplest infrastructure and recover higher concentration of EV than standard protocol. A workflow from sampling to characterization of EVs is proposed.

  17. Intercomparison of analysis methods for seismically isolated nuclear structures. Part 1: Advanced test data and numerical methods. Working material

    International Nuclear Information System (INIS)

    1993-01-01

    The purpose of the meeting was to review proposed contributions from CRP participating organizations to discuss in detail the experimental data on seismic isolators, to review the numerical methods for the analysis of the seismic isolators, and to perform a first comparison of the calculation results. The aim of the CRP was to validate the reliable numerical methods used for both detailed evaluation of dynamic behaviour of isolation devices and isolated nuclear structures of different nuclear power plant types. The full maturity of seismic isolation for nuclear applications was stressed, as well as the excellent behaviour of isolated structures during the recent earthquakes in Japan and the USA. Participants from Italy, USA, Japan, Russian federation, Republic of Korea, United Kingdom, India and European Commission have presented overview papers on the present programs and their status of contribution to the CRP

  18. Intercomparison of analysis methods for seismically isolated nuclear structures. Part 1: Advanced test data and numerical methods. Working material

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1993-07-01

    The purpose of the meeting was to review proposed contributions from CRP participating organizations to discuss in detail the experimental data on seismic isolators, to review the numerical methods for the analysis of the seismic isolators, and to perform a first comparison of the calculation results. The aim of the CRP was to validate the reliable numerical methods used for both detailed evaluation of dynamic behaviour of isolation devices and isolated nuclear structures of different nuclear power plant types. The full maturity of seismic isolation for nuclear applications was stressed, as well as the excellent behaviour of isolated structures during the recent earthquakes in Japan and the USA. Participants from Italy, USA, Japan, Russian federation, Republic of Korea, United Kingdom, India and European Commission have presented overview papers on the present programs and their status of contribution to the CRP.

  19. Effect of starch isolation method on properties of sweet potato starch

    Directory of Open Access Journals (Sweden)

    A. SURENDRA BABU

    2014-08-01

    Full Text Available Isolation method of starch with different agents influences starch properties, which provide attention for studying the most appropriate method for isolation of starch. In the present study sweet potato starch was isolated by Sodium metabisulphate (M1, Sodium chloride (M2, and Distilled water (M3 methods and these were assessed for functional, chemical, pasting and structural properties. M3 yielded the greatest recovery of starch (10.20%. Isolation methods significantly changed swelling power and pasting properties but starches exhibited similar chemical properties. Sweet potato starches possessed C-type diffraction pattern. Small size granules of 2.90 μm were noticed in SEM of M3 starch. A high degree positive correlation was found between ash, amylose, and total starch content. The study concluded that isolation methods brought changes in yield, pasting and structural properties of sweet potato starch.

  20. How the RNA isolation method can affect microRNA microarray results

    DEFF Research Database (Denmark)

    Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas

    2011-01-01

    RNA microarray analysis on porcine brain tissue. One method is a phenol-guanidine isothiocyanate-based procedure that permits isolation of total RNA. The second method, miRVana™ microRNA isolation, is column based and recovers the small RNA fraction alone. We found that microarray analyses give different results...... that depend on the RNA fraction used, in particular because some microRNAs appear very sensitive to the RNA isolation method. We conclude that precautions need to be taken when comparing microarray studies based on RNA isolated with different methods.......The quality of RNA is crucial in gene expression experiments. RNA degradation interferes in the measurement of gene expression, and in this context, microRNA quantification can lead to an incorrect estimation. In the present study, two different RNA isolation methods were used to perform micro...

  1. Methods for isolation and viability assessment of biological organisms

    Science.gov (United States)

    Letant, Sonia Edith; Baker, Sarah Elyse; Bond, Tiziana; Chang, Allan Shih-Ping

    2015-02-03

    Isolation of biological or chemical organisms can be accomplished using a surface enhanced Raman scattering (SERS) system. The SERS system can be a single or a stacked plurality of photonic crystal membranes with noble-metal lined through pores for flowing analyte potentially containing the biological or chemical organisms. The through pores can be adapted to trap individual biological or chemical organisms and emit SERS spectra, which can then be detected by a detector and further analyzed for viability of the biological or chemical organism.

  2. A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

    International Nuclear Information System (INIS)

    Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

    2005-01-01

    Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

  3. Stress and Deformation Analysis in Base Isolation Elements Using the Finite Element Method

    Directory of Open Access Journals (Sweden)

    Claudiu Iavornic

    2011-01-01

    Full Text Available In Modern tools as Finite Element Method can be used to study the behavior of elastomeric isolation systems. The simulation results obtained in this way provide a large series of data about the behavior of elastomeric isolation bearings under different types of loads and help in taking right decisions regarding geometrical optimizations needed for improve such kind of devices.

  4. Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content.

    Science.gov (United States)

    Laukkanen, R; Hakkinen, M; Lundén, J; Fredriksson-Ahomaa, M; Johansson, T; Korkeala, H

    2010-03-01

    The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. The four methods comprised of 15 isolation steps using selective enrichments (irgasan-ticarcillin-potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25 degrees C. Salmonella-Shigella-desoxycholate-calcium chloride agar, cefsulodin-irgasan-novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre-enrichment step with further selective enrichment showed the highest sensitivities (55-66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.

  5. Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

    Science.gov (United States)

    Kinashi, Haruyasu

    2011-01-01

    Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

  6. Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus▿

    Science.gov (United States)

    Stevens, D. Cole; Henry, Michael R.; Murphy, Kimberly A.; Boddy, Christopher N.

    2010-01-01

    New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters. PMID:20208031

  7. Method for the detection and isolation of traces of organic fluorine compounds in plants

    Energy Technology Data Exchange (ETDEWEB)

    Wade, R H; Ross, J M; Benedict, H M

    1964-01-01

    A method for the detection and isolation of sub-microgram quantities of organic fluorine compounds from plant materials in the presence of much larger amounts of inorganic fluoride is presented. The procedure consists first of a rapid screening step for use with large numbers of vegetable samples and extracts and, second, of a chromatographic step to isolate and characterize any fluoro-organics found. These methods are developed in light of specific chemical characteristics of organic fluorine compounds as a general class. A modification of SOEP's quantitative sub-micro fluoride analytical method is presented as applicable to these isolation methods. Microgram quantities of organic fluorine compounds were found in the plant materials investigated but at a level too low for isolation and identification.

  8. An Improved Quick Method for the Isolation of Total RNA from Cotton ...

    African Journals Online (AJOL)

    David PANG

    2011-11-02

    Nov 2, 2011 ... in liquid nitrogen in a mortar and pestle and stored until. RNA isolation. ... our laboratory for microarray analysis, cDNA pyro- sequencing studies and construction ..... Economic and rapid method for extracting cotton genomic ...

  9. Metabolic engineering of biosynthetic pathway for production of renewable biofuels.

    Science.gov (United States)

    Singh, Vijai; Mani, Indra; Chaudhary, Dharmendra Kumar; Dhar, Pawan Kumar

    2014-02-01

    Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

  10. Base Isolation for Seismic Retrofitting of a Multiple Building Structure: Evaluation of Equivalent Linearization Method

    Directory of Open Access Journals (Sweden)

    Massimiliano Ferraioli

    2016-01-01

    Full Text Available Although the most commonly used isolation systems exhibit nonlinear inelastic behaviour, the equivalent linear elastic analysis is commonly used in the design and assessment of seismic-isolated structures. The paper investigates if the linear elastic model is suitable for the analysis of a seismically isolated multiple building structure. To this aim, its computed responses were compared with those calculated by nonlinear dynamic analysis. A common base isolation plane connects the isolation bearings supporting the adjacent structures. In this situation, the conventional equivalent linear elastic analysis may have some problems of accuracy because this method is calibrated on single base-isolated structures. Moreover, the torsional characteristics of the combined system are significantly different from those of separate isolated buildings. A number of numerical simulations and parametric studies under earthquake excitations were performed. The accuracy of the dynamic response obtained by the equivalent linear elastic model was calculated by the magnitude of the error with respect to the corresponding response considering the nonlinear behaviour of the isolation system. The maximum displacements at the isolation level, the maximum interstorey drifts, and the peak absolute acceleration were selected as the most important response measures. The influence of mass eccentricity, torsion, and high-modes effects was finally investigated.

  11. Test of a mosquito eggshell isolation method and subsampling procedure.

    Science.gov (United States)

    Turner, P A; Streever, W J

    1997-03-01

    Production of Aedes vigilax, the common salt-marsh mosquito, can be assessed by determining eggshell densities found in soil. In this study, 14 field-collected eggshell samples were used to test a subsampling technique and compare eggshell counts obtained with a flotation method to those obtained by direct examination of sediment (DES). Relative precision of the subsampling technique was assessed by determining the minimum number of subsamples required to estimate the true mean and confidence interval of a sample at a predetermined confidence level. A regression line was fitted to cube-root transformed eggshell counts obtained from flotation and DES and found to be significant (P eggshells present. Eggshells obtained with the flotation method can be used to predict those from DES using the following equation: DES count = [1.386 x (flotation count)0.33 - 0.01]3.

  12. Extraction methods of Amaranthus sp. grain oil isolation.

    Science.gov (United States)

    Krulj, Jelena; Brlek, Tea; Pezo, Lato; Brkljača, Jovana; Popović, Sanja; Zeković, Zoran; Bodroža Solarov, Marija

    2016-08-01

    Amaranthus sp. is a fast-growing crop with well-known beneficial nutritional values (rich in protein, fat, dietary fiber, ash, and minerals, especially calcium and sodium, and containing a higher amount of lysine than conventional cereals). Amaranthus sp. is an underexploited plant source of squalene, a compound of high importance in the food, cosmetic and pharmaceutical industries. This paper has examined the effects of the different extraction methods (Soxhlet, supercritical fluid and accelerated solvent extraction) on the oil and squalene yield of three genotypes of Amaranthus sp. grain. The highest yield of the extracted oil (78.1 g kg(-1) ) and squalene (4.7 g kg(-1) ) in grain was obtained by accelerated solvent extraction (ASE) in genotype 16. Post hoc Tukey's HSD test at 95% confidence limit showed significant differences between observed samples. Principal component analysis (PCA) and cluster analysis (CA) were used for assessing the effect of different genotypes and extraction methods on oil and squalene yield, and also the fatty acid composition profile. Using coupled PCA and CA of observed samples, possible directions for improving the quality of product can be realized. The results of this study indicate that it is very important to choose both the right genotype and the right method of extraction for optimal oil and squalene yield. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  13. A rapid and low-cost DNA extraction method for isolating ...

    African Journals Online (AJOL)

    The price of commercial DNA extraction methods makes the routine use of polymerase chain reaction amplification (PCR) based methods rather costly for scientists in developing countries. A guanidium thiocayante-based DNA extraction method was investigated in this study for the isolation of Escherichia coli (E. coli) DNA ...

  14. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively...... affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact...

  15. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts.

    Science.gov (United States)

    Nielsen, Agnieszka Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos; Wlodarczyk, Artur Jacek; Gnanasekaran, Thiyagarajan; Perestrello Ramos H de Jesus, Maria; King, Brian Christopher; Bakowski, Kamil; Jensen, Poul Erik

    2016-07-01

    Chloroplasts in plants and algae and photosynthetic microorganisms such as cyanobacteria are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals and complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression of the appropriate pathways, but this requires optimization of carbon flux and reducing power, and a thorough understanding of regulatory pathways. Secretion or storage of the compounds produced can be exploited for the isolation or confinement of the desired compounds. In this review, we explore the use of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the levels of production to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons derived from the photosynthetic light reactions, appears to be non-limiting, but redirection of the fixed carbon via precursor molecules presents a challenge. We also discuss the available synthetic biology tools and the need to expand the molecular toolbox to facilitate cellular reprogramming for increased production yields in both cyanobacteria and chloroplasts. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  16. Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells

    Science.gov (United States)

    2017-02-01

    a cell population particularly suitable for low serum propagation, provided that appropriate growth factors are available. A low serum medium...of MGK. 15. SUBJECT TERMS Cornea, chemical warfare agent, corneal endothelial cell, endothelium, growth , isolation, mouse, rabbit, porcine, in...with corneal SM exposure.2 A primary requirement in achieving this goal is to develop methods that enable the isolation of a pure CEC population and

  17. Evaluation of four methods for estimating leaf area of isolated trees

    Science.gov (United States)

    P.J. Peper; E.G. McPherson

    2003-01-01

    The accurate modeling of the physiological and functional processes of urban forests requires information on the leaf area of urban tree species. Several non-destructive, indirect leaf area sampling methods have shown good performance for homogenous canopies. These methods have not been evaluated for use in urban settings where trees are typically isolated and...

  18. Non liquid nitrogen-based-method for isolation of DNA from ...

    African Journals Online (AJOL)

    A simple, efficient, reliable and cost-effective method for isolation of total genomic DNA from fungi, suitable for polymerase chain reaction (PCR) amplification and other molecular applications was described. The main advantages of the method are: (1) does not require the use of liquid nitrogen for preparation of fungi DNA; ...

  19. Antifungal susceptibility testing of vaginal candida isolates: the broth microdilution method

    Directory of Open Access Journals (Sweden)

    Mahmoudi Rad M

    2010-02-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Vulvovaginal candidiasis is a common mucosal infection among immunocompetent, healthy women, and is caused by opportunistic yeasts that belong to genus Candida. In this study, we isolated and identified the Candida species in the vagina of patients who admitted in Gynecology Department of Mahdieh Hospital in Tehran, Iran to evaluate the in vitro activities of fluconazole, miconazole, itraconazole and flucytosine against 191 clinical Candida isolates by the NCCLS microdilution method."n"nMethods: 191 Candida were isolated from vaginal secretions and identified with conventional mycological methods in the diagnosis of Candida species. The identity of all strains was confirmed genotypically by multiplex PCR. In vitro susceptibility testing of vaginal Candida isolates was performed by the NCCLS broth microdilution method. The results were read at 48 h."n"nResults: Most C. albicans isolates (>90% were sensitive in vitro to the antifungal agents tested. Most C. glabrata isolates showed sensitivity to miconazole and then flucytosine while they were more resistant to Itraconazole and fluconazole. Many isolates of C. tropicalis were susceptible to miconazole and then fluconazole. They showed a little resistance to

  20. Comparison of the Histopaque-1119 method with the Plasmagel method for separation of blood leukocytes for cytomegalovirus isolation.

    Science.gov (United States)

    Slifkin, M; Cumbie, R

    1992-10-01

    Histopaque-1119 (Sigma Chemical Co., St. Louis, Mo.) and Plasmagel (Cellular Products, Inc., Buffalo, N.Y.) were compared as density gradient separation reagents for the separation of polymorphonuclear leukocytes and mononuclear cells from blood from the isolation of cytomegalovirus (CMV). Of 200 peripheral blood specimens examined, CMV was recovered from 51 by both methods. The time of detection of immunofluorescent sites or a cytopathic effect associated with CMV was similar by each method. The Histopaque-1119 method was less time-consuming than the Plasmagel method since it did not require a precentrifugation step for the settling of erythrocytes. The use of Histopaque-1119 will permit an effective alternative single-step method for the separation of blood leukocytes for the isolation of CMV.

  1. Remote vacuum or pressure sealing device and method for critical isolated systems

    Science.gov (United States)

    Brock, James David [Newport News, VA; Keith, Christopher D [Newport News, VA

    2012-07-10

    A remote vacuum or pressure sealing apparatus and method for making a radiation tolerant, remotely prepared seal that maintains a vacuum or pressure tight seal throughout a wide temperature range. The remote sealing apparatus includes a fixed threaded sealing surface on an isolated system, a gasket, and an insert consisting of a plug with a protruding sample holder. An insert coupling device, provided for inserting samples within the isolated system, includes a threaded fastener for cooperating with the fixed threaded sealing surface on the isolated system. The insert coupling device includes a locating pin for azimuthal orientation, coupling pins, a tooted coaxial socket wrench, and an insert coupling actuator for actuating the coupling pins. The remote aspect of the sealing apparatus maintains the isolation of the system from the user's environment, safely preserving the user and the system from detrimental effect from each respectively.

  2. A simple and efficient method for isolating small RNAs from different plant species

    Directory of Open Access Journals (Sweden)

    de Folter Stefan

    2011-02-01

    Full Text Available Abstract Background Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species. Results We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays. Conclusion Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays.

  3. Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

    Directory of Open Access Journals (Sweden)

    Pricilla DM de Matos

    2010-11-01

    Full Text Available A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD, agar dilution (AD, latex agglutination (LA, oxacillin agar screening (OAS and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS-98.3% (AD. The LA test showed the second lowest sensitivity (86.4%. The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

  4. Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types.

    Science.gov (United States)

    Matos, Pricilla D M de; Schuenck, Ricardo P; Cavalcante, Fernanda S; Caboclo, Roberta M F; Santos, Kátia Regina N dos

    2010-11-01

    A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

  5. Modified method for combined DNA and RNA isolation from peanut and other oil seeds.

    Science.gov (United States)

    Dang, Phat M; Chen, Charles Y

    2013-02-01

    Isolation of good quality RNA and DNA from seeds is difficult due to high levels of polysaccharides, polyphenols, and lipids that can degrade or co-precipitate with nucleic acids. Standard RNA extraction methods utilizing guanidinium-phenol-chloroform extraction has not shown to be successful. RNA isolation from plant seeds is a prerequisite for many seed specific gene expression studies and DNA is necessary in marker-assisted selection and other genetic studies. We describe a modified method to isolate both RNA and DNA from the same seed tissue and have been successful with several oil seeds including peanut, soybean, sunflower, canola, and oil radish. An additional LiCl precipitation step was added to isolate both RNA and DNA from the same seed tissues. High quality nucleic acids were observed based on A(260)/A(280) and A(260)/A(230) ratios above 2.0 and distinct bands on gel-electrophoresis. RNA was shown to be suitable for reverse transcriptase polymerase chain reaction based on actin or 60S ribosomal primer amplification and DNA was shown to have a single band on gel-electrophoresis analysis. This result shows that RNA and DNA isolated using this method can be appropriate for molecular studies in peanut and other oil containing seeds.

  6. Comparison of the Histopaque-1119 method with the Plasmagel method for separation of blood leukocytes for cytomegalovirus isolation.

    OpenAIRE

    Slifkin, M; Cumbie, R

    1992-01-01

    Histopaque-1119 (Sigma Chemical Co., St. Louis, Mo.) and Plasmagel (Cellular Products, Inc., Buffalo, N.Y.) were compared as density gradient separation reagents for the separation of polymorphonuclear leukocytes and mononuclear cells from blood from the isolation of cytomegalovirus (CMV). Of 200 peripheral blood specimens examined, CMV was recovered from 51 by both methods. The time of detection of immunofluorescent sites or a cytopathic effect associated with CMV was similar by each method....

  7. A combined dynamic analysis method for geometrically nonlinear vibration isolators with elastic rings

    Science.gov (United States)

    Hu, Zhan; Zheng, Gangtie

    2016-08-01

    A combined analysis method is developed in the present paper for studying the dynamic properties of a type of geometrically nonlinear vibration isolator, which is composed of push-pull configuration rings. This method combines the geometrically nonlinear theory of curved beams and the Harmonic Balance Method to overcome the difficulty in calculating the vibration and vibration transmissibility under large deformations of the ring structure. Using the proposed method, nonlinear dynamic behaviors of this isolator, such as the lock situation due to the coulomb damping and the usual jump resulting from the nonlinear stiffness, can be investigated. Numerical solutions based on the primary harmonic balance are first verified by direct integration results. Then, the whole procedure of this combined analysis method is demonstrated and validated by slowly sinusoidal sweeping experiments with different amplitudes of the base excitation. Both numerical and experimental results indicate that this type of isolator behaves as a hardening spring with increasing amplitude of the base excitation, which makes it suitable for isolating both steady-state vibrations and transient shocks.

  8. Molecular evolution of the lysine biosynthetic pathways.

    Science.gov (United States)

    Velasco, A M; Leguina, J I; Lazcano, A

    2002-10-01

    Among the different biosynthetic pathways found in extant organisms, lysine biosynthesis is peculiar because it has two different anabolic routes. One is the diaminopimelic acid pathway (DAP), and the other over the a-aminoadipic acid route (AAA). A variant of the AAA route that includes some enzymes involved in arginine and leucine biosyntheses has been recently reported in Thermus thermophilus (Nishida et al. 1999). Here we describe the results of a detailed genomic analysis of each of the sequences involved in the two lysine anabolic routes, as well as of genes from other routes related to them. No evidence was found of an evolutionary relationship between the DAP and AAA enzymes. Our results suggest that the DAP pathway is related to arginine metabolism, since the lysC, asd, dapC, dapE, and lysA genes from lysine biosynthesis are related to the argB, argC, argD, argE, and speAC genes, respectively, whose products catalyze different steps in arginine metabolism. This work supports previous reports on the relationship between AAA gene products and some enzymes involved in leucine biosynthesis and the tricarboxylic acid cycle (Irvin and Bhattacharjee 1998; Miyazaki et al. 2001). Here we discuss the significance of the recent finding that several genes involved in the arginine (Arg) and leucine (Leu) biosynthesis participate in a new alternative route of the AAA pathway (Miyazaki et al. 2001). Our results demonstrate a clear relationship between the DAP and Arg routes, and between the AAA and Leu pathways.

  9. Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

    OpenAIRE

    Cirillo, J. D.; Weisbrod, T. R.; Banerjee, A.; Bloom, B. R.; Jacobs, W. R.

    1994-01-01

    The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the constru...

  10. Isolation and HPLC method development of azafrin from Alectra parasitica var. chitrakutensis.

    Science.gov (United States)

    Agrawal, Poonam; Laddha, Kirti; Tiwari, Ashok

    2014-01-01

    This study was undertaken to isolate and quantify azafrin in Alectra parasitica (Scrophulariaceae) rhizomes. A simple method for the isolation of carotenoid, azafrin, involves solvent extraction of the dried rhizome powder using a single solvent and further purification by recrystallisation. The structure of the compound was elucidated and confirmed by thin-layer chromatography, infrared spectroscopy, mass spectroscopy and nuclear magnetic resonance spectral analysis. A specific and rapid reversed-phase high-performance liquid chromatography (HPLC) method was developed for the analysis of azafrin. The method was validated for accuracy, precision, linearity and specificity. Validation revealed that the method is specific, accurate, precise, reliable and reproducible. The proposed HPLC method can be used for the identification and quantitative analysis of azafrin in A. parasitica rhizomes.

  11. Two methods for isolating the lung area of a CT scan for density information

    International Nuclear Information System (INIS)

    Hedlund, L.W.; Anderson, R.F.; Goulding, P.L.; Beck, J.W.; Effmann, E.L.; Putman, C.E.

    1982-01-01

    Extracting density information from irregularly shaped tissue areas of CT scans requires automated methods when many scans are involved. We describe two computer methods that automatically isolate the lung area of a CT scan. Each starts from a single, operator specified point in the lung. The first method follows the steep density gradient boundary between lung and adjacent tissues; this tracking method is useful for estimating the overall density and total area of lung in a scan because all pixels within the lung area are available for statistical sampling. The second method finds all contiguous pixels of lung that are within the CT number range of air to water and are not a part of strong density gradient edges; this method is useful for estimating density and area of the lung parenchyma. Structures within the lung area that are surrounded by strong density gradient edges, such as large blood vessels, airways and nodules, are excluded from the lung sample while lung areas with diffuse borders, such as an area of mild or moderate edema, are retained. Both methods were tested on scans from an animal model of pulmonary edema and were found to be effective in isolating normal and diseased lungs. These methods are also suitable for isolating other organ areas of CT scans that are bounded by density gradient edges

  12. Investigation of colistin sensitivity via three different methods in Acinetobacter baumannii isolates with multiple antibiotic resistance.

    Science.gov (United States)

    Sinirtaş, Melda; Akalin, Halis; Gedikoğlu, Suna

    2009-09-01

    In recent years there has been an increase in life-threatening infections caused by Acinetobacter baumannii with multiple antibiotic resistance, which has lead to the use of polymyxins, especially colistin, being reconsidered. The aim of this study was to investigate the colistin sensitivity of A. baumannii isolates with multiple antibiotic resistance via different methods, and to evaluate the disk diffusion method for colistin against multi-resistant Acinetobacter isolates, in comparison to the E-test and Phoenix system. The study was carried out on 100 strains of A. baumannii (colonization or infection) isolated from the microbiological samples of different patients followed in the clinics and intensive care units of Uludağ University Medical School between the years 2004 and 2005. Strains were identified and characterized for their antibiotic sensitivity by Phoenix system (Becton Dickinson, Sparks, MD, USA). In all studied A. baumannii strains, susceptibility to colistin was determined to be 100% with the disk diffusion, E-test, and broth microdilution methods. Results of the E-test and broth microdilution method, which are accepted as reference methods, were found to be 100% consistent with the results of the disk diffusion tests; no very major or major error was identified upon comparison of the tests. The sensitivity and the positive predictive value of the disk diffusion method were found to be 100%. Colistin resistance in A. baumannii was not detected in our region, and disk diffusion method results are in accordance with those of E-test and broth microdilution methods.

  13. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae (on linr)

    OpenAIRE

    Wang, Kui-Lin; Bolitho, Karen; Grafton, Karryn; Kortstee, A.J.; Karunairetnam, Sakuntala; McGhie, T.K.; Espley, R.V.; Hellens, R.P.; Allan, A.C.

    2010-01-01

    Background - The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all th...

  14. Carbapenem inactivation: a very affordable and highly specific method for phenotypic detection of carbapenemase-producing Pseudomonas aeruginosa isolates compared with other methods.

    Science.gov (United States)

    Akhi, Mohammad Taghi; Khalili, Younes; Ghotaslou, Reza; Kafil, Hossein Samadi; Yousefi, Saber; Nagili, Behroz; Goli, Hamid Reza

    2017-06-01

    This investigation was undertaken to compare phenotypic and molecular methods for detection of carbapenemase-producing Pseudomonas aeruginosa. A total of 245 non-duplicated isolates of P. aeruginosa were collected from hospitalized patients. Disc diffusion method was used to identify carbapenem-resistant bacteria. Three phenotypic methods, including Modified Hodge Test (MHT), Modified Carba NP (MCNP) test and Carbapenem Inactivation Method (CIM) were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was used to detect carbapenemase encoding genes. Of 245 P. aeruginosa isolates investigated, 121 isolates were carbapenem-resistant. Among carbapenem-resistant isolates, 40, 39 and 35 isolates exhibited positive results using MHT, MCNP test and CIM, respectively. PCR indicated the presence of carbapenemase genes in 35 of carbapenem-resistant isolates. MHT showed low sensitivity and specificity for carbapenemase detection among P. aeruginosa isolates in comparison to PCR. CIM was most affordable and highly specific than MCNP test compared with the molecular method.

  15. Enrichment Method for the Isolation of Bioactive Actinomycetes From Mangrove Sediments of Andaman Islands, India

    Directory of Open Access Journals (Sweden)

    Baskaran, R.

    2011-01-01

    Full Text Available Various pre-treatment methods and three different media were employed for the isolation of bioactive actinomycetes from mangrove sediments of Andaman and Nicobar Islands, India. Sediments from four different sites of mangrove forest were collected and pre-treated by dry heat method, and the media were supplemented with cycloheximide 80 µg/mL and nalidixic acid 75 µg/mL. The mean actinomycetes population density in sediment samples were recorded as 22 CFU-10^-6/gm in KUA medium followed by 12 CFU-10^-6/gm in AIA medium and 8 CFU-10^-6/gm in SCA medium. A total of 42 actinomycetes were isolated, and all the isolates were evaluated for their antibacterial activity against pathogenic bacteria on two different media. Among 42 isolates tested, 22 species were found to be antibacterial metabolite producer against test bacteria namely, Staphylococcus aureus, Bacillus subtilis, Salmonella typhi and Klebsiella pneumoniae. Particularly, the actinomycete strains such as A101, A102, A107, A116, A121, A125, A130, F101, F102, F104, F106, De101 and De102 significantly inhibited the growth of all bacteria which were tested. Of these strains, A107 was identified as Streptomyces spp. This strain had the maximum activity against all used pathogens on both medium. Hence, the isolation, characterization and studies of secondary metabolites of actinomycetes from mangrove sediments in Andaman and Nicobar Island could be a pathway for discovery of antibiotics from marine actinomycetes.

  16. A safe inexpensive method to isolate high quality plant and fungal ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-18

    Aug 18, 2008 ... quality DNA from plant and fungal species. This method uses potassium acetate to remove proteins and polysaccharides in an SDS extraction buffer. Further DNA purification is achieved using a low salt. CTAB treatment. This SDS/CTAB protocol was used to isolate high quality genomic DNA subject to.

  17. A safe inexpensive method to isolate high quality plant and fungal ...

    African Journals Online (AJOL)

    The most commonly used plant DNA isolation methods use toxic and hazardous chemicals (phenol, chloroform), which require special equipment to minimize exposure and may limit their use in certain environments. Commercial DNA extraction kits are convenient and usually safe, but their availability to certain developing ...

  18. A method for the isolation of root hairs from the model legume Medicago truncatula

    NARCIS (Netherlands)

    Ramos Escribano, J.; Bisseling, T.

    2003-01-01

    A new method for the isolation of root hairs from the model legume, Medicago truncatula, was developed. The procedure involves the propagation of detached roots on agar plates and the collection of root hairs by immersion in liquid nitrogen. Yields of up to 40 µg of root hair protein were obtained

  19. Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni

    DEFF Research Database (Denmark)

    Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst

    2017-01-01

    Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-formin...

  20. Isolated Liver Perfusion Using Percutaneous Methods:[ql An Experimental Study in the Pig

    International Nuclear Information System (INIS)

    Harnek, Jan; Cwikiel, Wojciech; Bergqvist, Lennart; Persson, Bo; Stridbeck, Hans

    1996-01-01

    Purpose: To develop a method for isolated perfusion of the liver using radiological methods. Methods: Twenty-one pigs, weighing about 20 kg, were divided into three groups. By transjugular and transfemoral approaches two occlusion balloons were placed in the inferior vena cava cranial and caudal, respectively, to the origin of the hepatic veins. One occlusion balloon was placed transfemorally in the common hepatic artery. Another occlusion balloon was inserted in the main branch of the portal vein via the transjugular-transhepatic approach in 11 pigs (groups 1 and 2), and in 10 pigs (group 3) by a percutaneous transhepatic route. After inflation of the balloons, patency of the isolated liver circulation was evaluated by recirculation of 99 Tc m -labelled human albumin during 30 min. Blood tests were obtained after 1, 3, 5, 10, 15, and 30 min to evaluate leakage from the liver to the systemic circulation. Results: Increasing leakage to the systemic circulation from the isolated liver circulation was observed in groups 1 and 2. In the third group the leakage was less than 10%. Conclusion: In an experimental animal model, isolated perfusion of the liver with minor leakage to the systemic circulation may be achieved using radiological methods

  1. Three methods for isolating viable anthozoan endoderm cells with their intracellular symbiotic dinoflagellates

    Science.gov (United States)

    Gates, R. D.; Muscatine, L.

    1992-09-01

    Three maceration methods are described for the isolation of single endoderm cells from marine cnidarians. Two are enzymatic treatments suitable for fleshy anthozoans such as sea anemones and zoanthids. The third employs calcium free sea water and is suitable for stony corals. The viability and morphology of the endoderm cells is described using fluorogenic dyes and scanning and transmission electron microscopy.

  2. An Overview of Neolignans of the Genus Piper L.: Isolation Methods and Biological Activities.

    Science.gov (United States)

    Macedo, Arthur Ladeira; Dos Santos, Thais Carvalho Costa; Valverde, Alessandra Leda; Moreira, Davyson de Lima; Vasconcelos, Thatyana Rocha Alves

    2017-01-01

    The genus Piper L. has the shikimic acid pathway predominantly expressed, biosynthesizing many cinnamic acid derivatives (CAD). Neolignans comprise an important class of CAD that exhibit a wide range of pharmacological properties such as antibacterial, antitumor, insecticidal, anti-inflammatory, antioxidant, smooth muscle relaxant, neuroprotective, antiprotozoal and against platelet aggregation factor. These substances have been extracted and isolated from Piper species using different technics. The present review aims to summarize extraction and isolation methods and biological activities of the different types of neolignans covering the period from 1968 to January 2016. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Impact of enumeration method on diversity of Escherichia coli genotypes isolated from surface water.

    Science.gov (United States)

    Martin, E C; Gentry, T J

    2016-11-01

    There are numerous regulatory-approved Escherichia coli enumeration methods, but it is not known whether differences in media composition and incubation conditions impact the diversity of E. coli populations detected by these methods. A study was conducted to determine if three standard water quality assessments, Colilert ® , USEPA Method 1603, (modified mTEC) and USEPA Method 1604 (MI), detect different populations of E. coli. Samples were collected from six watersheds and analysed using the three enumeration approaches followed by E. coli isolation and genotyping. Results indicated that the three methods generally produced similar enumeration data across the sites, although there were some differences on a site-by-site basis. The Colilert ® method consistently generated the least diverse collection of E. coli genotypes as compared to modified mTEC and MI, with those two methods being roughly equal to each other. Although the three media assessed in this study were designed to enumerate E. coli, the differences in the media composition, incubation temperature, and growth platform appear to have a strong selective influence on the populations of E. coli isolated. This study suggests that standardized methods of enumeration and isolation may be warranted if researchers intend to obtain individual E. coli isolates for further characterization. This study characterized the impact of three USEPA-approved Escherichia coli enumeration methods on observed E. coli population diversity in surface water samples. Results indicated that these methods produced similar E. coli enumeration data but were more variable in the diversity of E. coli genotypes observed. Although the three methods enumerate the same species, differences in media composition, growth platform, and incubation temperature likely contribute to the selection of different cultivable populations of E. coli, and thus caution should be used when implementing these methods interchangeably for

  4. 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.

  5. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Xu Guoqiang

    2012-02-01

    Full Text Available Abstract Background Fumaric acid is a commercially important component of foodstuffs, pharmaceuticals and industrial materials, yet the current methods of production are unsustainable and ecologically destructive. Results In this study, the fumarate biosynthetic pathway involving reductive reactions of the tricarboxylic acid cycle was exogenously introduced in S. cerevisiae by a series of simple genetic modifications. First, the Rhizopus oryzae genes for malate dehydrogenase (RoMDH and fumarase (RoFUM1 were heterologously expressed. Then, expression of the endogenous pyruvate carboxylase (PYC2 was up-regulated. The resultant yeast strain, FMME-001 ↑PYC2 + ↑RoMDH, was capable of producing significantly higher yields of fumarate in the glucose medium (3.18 ± 0.15 g liter-1 than the control strain FMME-001 empty vector. Conclusions The results presented here provide a novel strategy for fumarate biosynthesis, which represents an important advancement in producing high yields of fumarate in a sustainable and ecologically-friendly manner.

  6. Comparison of different DNA isolation methods and use of dodecyle trimethyl ammonium bromide (DTAB for the isolation of DNA from meat products

    Directory of Open Access Journals (Sweden)

    Yusuf OZsENSOY

    2016-12-01

    Conclusion: DNA isolation kit, another best method, is recommended due to quality and quantity of DNA for researchers who do not want that phenol/chloroform method have toxic substances. This study is also the first study in which DTAB method is used for DNA extraction from meat products. [J Adv Vet Anim Res 2016; 3(4.000: 368-374

  7. Development of an evaluation method for seismic isolation systems of nuclear power facilities. Seismic design analysis methods for crossover piping system

    International Nuclear Information System (INIS)

    Tai, Koichi; Sasajima, Keisuke; Fukushima, Shunsuke; Takamura, Noriyuki; Onishi, Shigenobu

    2014-01-01

    This paper provides seismic design analysis methods suitable for crossover piping system, which connects between seismic isolated building and non-isolated building in the seismic isolated nuclear power plant. Through the numerical study focused on the main steam crossover piping system, seismic response spectrum analysis applying ISM (Independent Support Motion) method with SRSS combination or CCFS (Cross-oscillator, Cross-Floor response Spectrum) method has found to be quite effective for the seismic design of multiply supported crossover piping system. (author)

  8. A 3-D aerodynamic method for the analysis of isolated horizontal-axis wind turbines

    Energy Technology Data Exchange (ETDEWEB)

    Ammara, I.; Masson, C.; Paraschivoiu, I. [Ecole Polytechnique, Montreal (Canada)

    1997-12-31

    In most existing performance-analysis methods, wind turbines are considered isolated so that interference effects caused by other rotors or by the site topography are neglected. The main objective of this paper is to propose a practical 3-D method suitable for the study of these effects, in order to optimize the arrangement and the positioning of Horizontal-Axis Wind Turbines (HAWTs) in a wind farm. In the proposed methodology, the flow field around isolated HAWTs is predicted by solving the 3-D, time-averaged, steady-state, incompressible, Navier-Stokes equations in which the turbines are represented by distributions of momentum sources. The resulting governing equations are solved using a Control-Volume Finite Element Method (CVFEM). The fundamental aspects related to the development of a practical 3-D method are discussed in this paper, with an emphasis on some of the challenges that arose during its implementation. The current implementation is limited to the analysis of isolated HAWTs. Preliminary results have indicated that, the proposed 3-D method reaches the same level of accuracy, in terms of performance predictions, that the previously developed 2-D axisymmetric model and the well-known momentum-strip theory, while still using reasonable computers resources. It can be considered as a useful tool for the design of HAWTs. Its main advantages, however, are its intrinsic capacity to predict the details of the flow in the wake, and its capabilities of modelling arbitrary wind-turbine arrangements and including ground effects.

  9. A highly efficient nonchemical method for isolating live nematodes (Caenorhabditis elegans) from soil during toxicity assays.

    Science.gov (United States)

    Kim, Shin Woong; Moon, Jongmin; An, Youn-Joo

    2015-01-01

    The success of soil toxicity tests using Caenorhabditis elegans may depend in large part on recovering the organisms from the soil. However, it can be difficult to learn the International Organization for Standardization/ASTM International recovery process that uses the colloidal silica flotation method. The present study determined that a soil-agar isolation method provides a highly efficient and less technically demanding alternative to the colloidal silica flotation method. Test soil containing C. elegans was arranged on an agar plate in a donut shape, a linear shape, or a C curve; and microbial food was placed outside the soil to encourage the nematodes to leave the soil. The effects of ventilation and the presence of food on nematode recovery were tested to determine the optimal conditions for recovery. A linear arrangement of soil on an agar plate that was sprinkled with microbial food produced nearly 83% and 90% recovery of live nematodes over a 3-h and a 24-h period, respectively, without subjecting the nematodes to chemical stress. The method was tested using copper (II) chloride dihydrate, and the resulting recovery rate was comparable to that obtained using colloidal silica flotation. The soil-agar isolation method portrayed in the present study enables live nematodes to be isolated with minimal additional physicochemical stress, making it a valuable option for use in subsequent sublethal tests where live nematodes are required. © 2014 SETAC.

  10. An intercomparison of three methods for the large-scale isolation of oceanic dissolved organic matter

    Science.gov (United States)

    Green, Nelson W.; Perdue, E. Michael; Aiken, George R.; Butler, Kenna D.; Chen, Hongmei; Dittmar, Thorsten; Niggemann, Jutta; Stubbins, Aron

    2014-01-01

    Dissolved organic matter (DOM) was isolated from large volumes of deep (674 m) and surface (21 m) ocean water via reverse osmosis/electrodialysis (RO/ED) and two solid-phase extraction (SPE) methods (XAD-8/4 and PPL) at the Natural Energy Laboratory of Hawaii Authority (NELHA). By applying the three methods to common water samples, the efficiencies of XAD, PPL and RO/ED DOM isolation were compared. XAD recovered 42% of dissolved organic carbon (DOC) from deep water (25% with XAD-8; 17% with XAD-4) and 30% from surface water (16% with XAD-8; 14% with XAD-4). PPL recovered 61 ± 3% of DOC from deep water and 61% from surface water. RO/ED recovered 82 ± 3% of DOC from deep water, 14 ± 3% of which was recovered in a sodium hydroxide rinse, and 75 ± 5% of DOC from surface water, with 12 ± 2% in the sodium hydroxide rinse. The highest recoveries of all were achieved by the sequential isolation of DOC, first with PPL and then via RO/ED. This combined technique recovered 98% of DOC from a deep water sample and 101% of DOC from a surface water sample. In total, 1.9, 10.3 and 1.6 g-C of DOC were collected via XAD, PPL and RO/ED, respectively. Rates of DOC recovery using the XAD, PPL and RO/ED methods were 10, 33 and 10 mg-C h− 1, respectively. Based upon C/N ratios, XAD isolates were heavily C-enriched compared with water column DOM, whereas RO/ED and PPL ➔ RO/ED isolate C/N values were most representative of the original DOM. All techniques are suitable for the isolation of large amounts of DOM with purities suitable for most advanced analytical techniques. Coupling PPL and RO/ED techniques may provide substantial progress in the search for a method to quantitatively isolate oceanic DOC, bringing the entirety of the DOM pool within the marine chemist's analytical window.

  11. Comparative miRNA Analysis of Urine Extracellular Vesicles Isolated through Five Different Methods

    Directory of Open Access Journals (Sweden)

    Felix Royo

    2016-12-01

    Full Text Available Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

  12. Fine typing of methicillin-resistant Staphylococcus aureus isolates using direct repeat unit and staphylococcal interspersed repeat unit typing methods.

    Science.gov (United States)

    Ho, Cheng-Mao; Ho, Mao-Wang; Li, Chi-Yuan; Lu, Jang-Jih

    2015-08-01

    Methicillin-resistant Staphylococcus aureus (MRSA) typing is an important epidemiologic tool for monitoring trends and preventing outbreaks. However, the efficiency of various MRSA typing methods for each SCCmec MRSA isolate is rarely evaluated. A total of 157 MRSA isolates from four different regions in Taiwan were typed with five different molecular methods, including SCCmec typing, multilocus sequence typing (MLST), spa typing, mec-associated direct repeat unit (dru) copy number determination, and staphylococcal interspersed repeat unit (SIRU) profiling. There were four SCCmec types, eight MLST types, 15 spa types, 11 dru types, and 31 SIRU profiles. The most common type determined by each molecular typing method was SCCmec III (115 isolates, 73.2%), ST239 (99 isolates, 63.1%), t037 (107 isolates, 68.2%), 14 dru copies (76 isolates, 48.4%), and SIRU profile 3013722 (102 isolates, 65%), respectively. When using the combination of MLST, spa typing, and dru copy number, ST5-t002-4 (n = 8), ST239-t037-14 (n = 68), ST59-t437-9 (n = 9), and ST59-t437-11 (n = 6) were found to be the most common types of SCCmec types II (n = 9), III (n = 115), IV (n = 21), and VT (n = 11) isolates, respectively. SCCmec type III isolates were further classified into 11 dru types. Of the 21 SCCmec type IV isolates, 14 SIRU profiles were found. Seven SIRU patterns were observed in the 11 SCCmec type VT isolates. Different typing methods showed a similar Hunter-Gaston discrimination index among the 157 MRSA isolates. However, dru and SIRU typing methods had a better discriminatory power for SCCmec type III and SCCmec types IV and VT isolates, respectively, suggesting that dru and SIRU can be used to further type these isolates. Copyright © 2013. Published by Elsevier B.V.

  13. Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria.

    Science.gov (United States)

    Dittmann, Elke; Gugger, Muriel; Sivonen, Kaarina; Fewer, David P

    2015-10-01

    Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with intricate chemical structures and potent biological activities. The bulk of these natural products are known from just a handful of genera. Recent efforts have elucidated the mechanisms underpinning the biosynthesis of a diverse array of natural products from cyanobacteria. Many of the biosynthetic mechanisms are unique to cyanobacteria or rarely described from other organisms. Advances in genome sequence technology have precipitated a deluge of genome sequences for cyanobacteria. This makes it possible to link known natural products to biosynthetic gene clusters but also accelerates the discovery of new natural products through genome mining. These studies demonstrate that cyanobacteria encode a huge variety of cryptic gene clusters for the production of natural products, and the known chemical diversity is likely to be just a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms. Copyright © 2015. Published by Elsevier Ltd.

  14. Comparative studies of two methods for miRNA isolation from milk whey.

    Science.gov (United States)

    Jin, Xiao-lu; Wei, Zi-hai; Liu, Lan; Liu, Hong-yun; Liu, Jian-xin

    2015-06-01

    MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS(®) followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS(®) followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100).

  15. Comparative studies of two methods for miRNA isolation from milk whey*

    Science.gov (United States)

    Jin, Xiao-lu; Wei, Zi-hai; Liu, Lan; Liu, Hong-yun; Liu, Jian-xin

    2015-01-01

    MicroRNAs (miRNAs) from milk whey have been considered for their potential as noninvasive biomarkers for milk quality control and disease diagnosis. However, standard protocols for miRNA isolation and quantification from milk whey are not well established. The objective of this study was to compare two methods for the isolation of miRNAs from milk whey. These two methods were modified phenol-based technique (Trizol LS® followed by phenol precipitation, the TP method) and combined phenol and column-based approach (Trizol LS® followed by cleanup using the miRNeasy kit, the TM method). Yield and quality of RNA were rigorously measured using a NanoDrop ND-1000 spectrophotometer and then the distribution of RNA was precisely detected in a Bioanalyzer 2100 instrument by microchip gel electrophoresis. Several endogenous miRNAs (bta-miR-141, bta-miR-146a, bta-miR-148a, bta-miR-200c, bta-miR-362, and bta-miR-375) and an exogenous spike-in synthetic control miRNA (cel-miR-39) were quantified by real-time polymerase chain reaction (PCR) to examine the apparent recovery efficiency of milk whey miRNAs. Both methods could successfully isolate sufficient small RNA (whey, and their yields were quite similar. However, the quantification results show that the total miRNA recovery efficiency by the TM method is superior to that by the TP method. The TM method performed better than the TP for recovery of milk whey miRNA due to its consistency and good repeatability in endogenous and spike-in miRNA recovery. Additionally, quantitative recovery analysis of a spike-in miRNA may be more accurate to reflect the milk whey miRNA recovery efficiency than using traditional RNA quality analysis instruments (NanoDrop or Bioanalyzer 2100). PMID:26055915

  16. Evolutionary systems biology of amino acid biosynthetic cost in yeast.

    Directory of Open Access Journals (Sweden)

    Michael D Barton

    2010-08-01

    Full Text Available Every protein has a biosynthetic cost to the cell based on the synthesis of its constituent amino acids. In order to optimise growth and reproduction, natural selection is expected, where possible, to favour the use of proteins whose constituents are cheaper to produce, as reduced biosynthetic cost may confer a fitness advantage to the organism. Quantifying the cost of amino acid biosynthesis presents challenges, since energetic requirements may change across different cellular and environmental conditions. We developed a systems biology approach to estimate the cost of amino acid synthesis based on genome-scale metabolic models and investigated the effects of the cost of amino acid synthesis on Saccharomyces cerevisiae gene expression and protein evolution. First, we used our two new and six previously reported measures of amino acid cost in conjunction with codon usage bias, tRNA gene number and atomic composition to identify which of these factors best predict transcript and protein levels. Second, we compared amino acid cost with rates of amino acid substitution across four species in the genus Saccharomyces. Regardless of which cost measure is used, amino acid biosynthetic cost is weakly associated with transcript and protein levels. In contrast, we find that biosynthetic cost and amino acid substitution rates show a negative correlation, but for only a subset of cost measures. In the economy of the yeast cell, we find that the cost of amino acid synthesis plays a limited role in shaping transcript and protein expression levels compared to that of translational optimisation. Biosynthetic cost does, however, appear to affect rates of amino acid evolution in Saccharomyces, suggesting that expensive amino acids may only be used when they have specific structural or functional roles in protein sequences. However, as there appears to be no single currency to compute the cost of amino acid synthesis across all cellular and environmental

  17. [Means and methods of personal hygiene in the experiment with 520-day isolation].

    Science.gov (United States)

    Shumilina, G A; Shumilina, I V; Solov'eva, S O

    2013-01-01

    Six volunteers (3 Russians, a Frenchman, an Italian and a Chinese) participated in assessment of the input of sanitation and housekeeping provisions to their wellbeing during 520-day isolation and confinement. Subject of the study was quality and sufficiency of housekeeping agents and procedures as well as more than 60 names of personal hygiene items. The sanitation and housekeeping monitoring involved the clinical, hygienic and microbiological methods, and also consideration of crew comments on the items at their disposal and recommended procedures. Based on the analysis of the functional condition of the integument and oral cavity and entries in the questionnaires, i.e. objective data and subjective feelings, all test subjects remained in the invariably good state. Owing to the application of the selected hygienic means and methods the microbial status of the crew was stable throughout 520-day isolation.

  18. Comparison of Plasmagel with LeucoPREP-Macrodex methods for separation of leukocytes for virus isolation.

    Science.gov (United States)

    Woods, G L; Proffitt, M R

    1987-10-01

    Plasmagel (Cellular Products, Inc., Buffalo, NY), which can separate both polymorphonuclear leukocytes (PMN) and mononuclear cells from other blood components, and LeucoPREP (Becton Dickinson Immunocytometry Systems, Mountain View, CA), which can separate mononuclear cells from other blood components, were used to harvest leukocytes from whole blood for the purpose of virus isolation. Macrodex was combined with the later, in a second step, for recovery of PMN. Of 90 peripheral blood specimens examined, cytomegalovirus was recovered from 10: in six by both methods, in three from Plasmagel prepared cells only, and in one from cells from the LeucoPREP-Macrodex preparation only. Total leukocyte counts, differential counts, and leukocyte viability did not differ significantly for the two methods. Plasmagel provided an efficient, inexpensive means of harvesting leukocytes from whole blood for virus isolation.

  19. Improved Method for the Isolation of Biosurfactant Glycolipids from Rhodococcus sp. Strain H13A

    OpenAIRE

    Bryant, Frank O.

    1990-01-01

    An improved method for the isolation of the biosurfactant glycolipids from Rhodococcus sp. strain H13A by using XM 50 diafiltration and isopropanol precipitation was devised. This procedure was advantageous since it removes protein coisolated when the glycolipids are obtained by organic extraction and silicic acid chromatography. The protein apparently does not contribute any biosurfactant characteristics to the glycolipids. The deacylated glycolipid backbone included only a disaccharide.

  20. Sensitive microculture method for isolation of human immunodeficiency virus type 1 from blood leukocytes.

    Science.gov (United States)

    Erice, A; Sannerud, K J; Leske, V L; Aeppli, D; Balfour, H H

    1992-02-01

    A study was conducted to compare our standard culture with a new microculture procedure for isolation of human immunodeficiency virus type 1 (HIV-1) from blood leukocytes. A total of 137 blood specimens from 102 HIV-1 antibody-positive individuals (52 were asymptomatic, 31 were symptomatic, and 19 had AIDS) were cultured in a microculture system in which 10(6) of the patients' peripheral blood mononuclear cells (PBMC) were cocultured with 10(6) phytohemagglutinin (PHA)-stimulated PBMC from an HIV-1 antibody-negative blood donor in 1.2 ml of culture medium. Results were compared with those of a historical control group of 139 standard HIV-1 cultures from 108 HIV-1 antibody-positive subjects (58 were asymptomatic, 36 were symptomatic, and 14 had AIDS). For standard cultures, 10 x 10(6) of the patients' PBMC were cocultured with 5 x 10(6) PHA-stimulated PBMC from an HIV-1 antibody-negative blood donor in 15 ml of culture medium. HIV-1 was isolated in 128 (93%) microcultures and 133 (96%) standard cultures. Both methods identified more than 75% of the positive cultures within 7 days and 100% of the positive cultures within 14 days. The isolation rates for HIV-1 in microcultures compared with standard cultures were 91 versus 93% (specimens from asymptomatic individuals), 93 versus 96% (specimens from symptomatic individuals), and 97 versus 100% (specimens from patients with AIDS). The median time to positivity for both culture methods was 7 days, and this correlated significantly with symptoms and CD4+ cell counts. The microculture method is a sensitive and less expensive system for isolation of HIV-1 from PBMC of HIV-1 antibody-positive individuals, and we recommend it as the culture method of choice, especially for children and patients with AIDS and severe anemia or leukopenia whose blood volume is an important consideration.

  1. Contribution of the JRC Ispra to the intercomparison of analysis methods for seismically isolated nuclear structures

    International Nuclear Information System (INIS)

    Magonette, G.; Renda, V.

    2002-01-01

    Aim of the work done at JRC has been essentially to investigate the potentiality of the Pseudo-Dynamic (PsD) method to test structures incorporating anti-seismic protection devices based on materials with a strain-rate dependent behaviour. This is of relevant importance due to the interest to perform tests on large-scale mock-ups to assess the behaviour of realistic structure of civil engineering interest. Two specific typologies of protection have been analysed and tested at the European Laboratory for Structural Assessment (ELSA) of JRC Ispra. The first dealing with base isolation and the second with energy dissipation devices. In both cases the protection devices were based on high damping rubber material which is characterised by a moderate dependence from the strain rate of the application of the displacements. To validate a standard procedure to test base isolated structures by the PsD method, a collaboration was set up with the Italian Working Group on Seismic Isolation which includes the national research centre ENEA, the national electricity board ENEL, the industrial research centre ISMES and a manufacturer of isolators ALGA. In the framework of this collaboration it was decided to test at the ELSA laboratory a scaled 5-storey frame structure (provided by ENEL), isolated by means of high damping rubber bearings (HDRBs), which had been tested on the shaking table of ISMES. This experimental activity aimed to compare the results which can be obtained by means of the PsD testing technique with those which can be obtained by means of a truly-dynamic test on a shaking table. To validate a standard procedure to test structures incorporating energy dissipation devices, an international collaboration has been set up with Industries, Research Centres and Universities in the framework of a project partially funded by the European Commission through the General Directorate for Science and Technology. The obtained results show once more that the PsD method, when

  2. A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid.

    Science.gov (United States)

    Holewinski, Ronald J; Jin, Zhicheng; Powell, Matthew J; Maust, Matthew D; Van Eyk, Jennifer E

    2013-03-01

    Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high-abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC-MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti-HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production

    Directory of Open Access Journals (Sweden)

    Juwairiah Remali

    2017-11-01

    Full Text Available Background Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy carbonyl phenazine-1-carboxylic acid (HCPCA extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea. Methods The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites. Results The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35% consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis. Discussion The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites.

  4. Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Han, Li; Huang, Xueshi [Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819 (China); He, Jing, E-mail: hejingjj@mail.hzau.edu.cn [National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China)

    2016-04-22

    Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

  5. Integration of Fermentation and Organic Synthesis: Studies of Roquefortine C and Biosynthetic Derivatives

    Science.gov (United States)

    Gober, Claire Marie

    Roquefortine C is one of the most ubiquitous indoline alkaloids of fungal origin. It has been isolated from over 30 different species of Penicillium fungi and has garnered attention in recent years for its role as a biosynthetic precursor to the triazaspirocyclic natural products glandicoline B, meleagrin, and oxaline. The triazaspirocyclic motif, which encompasses three nitrogen atoms attached to one quaternary carbon forming a spirocyclic scaffold, is a unique chemical moiety that has been shown to impart a wide array of biological activity, from anti-bacterial activity and antiproliferative activity against cancer cell lines to anti-biofouling against marine organisms. Despite the promise of these compounds in the pharmaceutical and materials industries, few syntheses of triazaspirocycles exist in the literature. The biosynthesis of roquefortine C-derived triazaspirocycles, however, provides inspiration for the synthesis of these compounds, namely through a nitrone-promoted transannular rearrangement. This type of internal rearrangement has never been carried out synthetically and would provide an efficient stereoselective synthesis of triazaspirocycles. This work encompasses efforts towards elucidating the biosynthetic pathway of roquefortine C-derived triazaspirocycles as well as synthetic efforts towards the construction of triazaspirocycles. Chapter 1 will discuss a large-scale fermentation procedure for the production of roquefortine C from Penicillium crustosum. Chapters 2 and 3 explore (through enzymatic and synthetic means, respectively) the formation of the key indoline nitrone moiety required for the proposed transannular rearrangement. Finally, chapter 4 will discuss synthetic efforts towards the synthesis of triazaspirocycles. This work has considerably enhanced our understanding of the roquefortine C biosynthetic pathway and the unique chemistry of this natural product, and our efforts towards the synthesis of triazaspirocycles will facilitate the

  6. Identification of an unusual type II thioesterase in the dithiolopyrrolone antibiotics biosynthetic pathway

    International Nuclear Information System (INIS)

    Zhai, Ying; Bai, Silei; Liu, Jingjing; Yang, Liyuan; Han, Li; Huang, Xueshi; He, Jing

    2016-01-01

    Dithiolopyrrolone group antibiotics characterized by an electronically unique dithiolopyrrolone heterobicyclic core are known for their antibacterial, antifungal, insecticidal and antitumor activities. Recently the biosynthetic gene clusters for two dithiolopyrrolone compounds, holomycin and thiomarinol, have been identified respectively in different bacterial species. Here, we report a novel dithiolopyrrolone biosynthetic gene cluster (aut) isolated from Streptomyces thioluteus DSM 40027 which produces two pyrrothine derivatives, aureothricin and thiolutin. By comparison with other characterized dithiolopyrrolone clusters, eight genes in the aut cluster were verified to be responsible for the assembly of dithiolopyrrolone core. The aut cluster was further confirmed by heterologous expression and in-frame gene deletion experiments. Intriguingly, we found that the heterogenetic thioesterase HlmK derived from the holomycin (hlm) gene cluster in Streptomyces clavuligerus significantly improved heterologous biosynthesis of dithiolopyrrolones in Streptomyces albus through coexpression with the aut cluster. In the previous studies, HlmK was considered invalid because it has a Ser to Gly point mutation within the canonical Ser-His-Asp catalytic triad of thioesterases. However, gene inactivation and complementation experiments in our study unequivocally demonstrated that HlmK is an active distinctive type II thioesterase that plays a beneficial role in dithiolopyrrolone biosynthesis. - Highlights: • Cloning of the aureothricin biosynthetic gene cluster from Streptomyces thioluteus DSM 40027. • Identification of the aureothricin gene cluster by heterologous expression and in-frame gene deletion. • The heterogenetic thioesterase HlmK significantly improved dithiolopyrrolones production of the aureothricin gene cluster. • Identification of HlmK as an unusual type II thioesterase.

  7. IMG-ABC: An Atlas of Biosynthetic Gene Clusters to Fuel the Discovery of Novel Secondary Metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Chen, I-Min; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Huang, Jinghua; Reddy, T. B.K.; Cimermancic, Peter; Fischbach, Michael; Ivanova, Natalia; Markowitz, Victor; Kyrpides, Nikos; Pati, Amrita

    2014-10-28

    In the discovery of secondary metabolites (SMs), large-scale analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of relevant computational resources. We present IMG-ABC (https://img.jgi.doe.gov/abc/) -- An Atlas of Biosynthetic gene Clusters within the Integrated Microbial Genomes (IMG) system1. IMG-ABC is a rich repository of both validated and predicted biosynthetic clusters (BCs) in cultured isolates, single-cells and metagenomes linked with the SM chemicals they produce and enhanced with focused analysis tools within IMG. The underlying scalable framework enables traversal of phylogenetic dark matter and chemical structure space -- serving as a doorway to a new era in the discovery of novel molecules.

  8. A method for starting up the gas isolating section of a benzine pyrolysis installation

    Energy Technology Data Exchange (ETDEWEB)

    Bruskin, Yu.A.; Gorokhov, V.V.; Kotler, L.D.; Shevchenko, K.N.; Zeldin, V.Ye.

    1982-01-01

    In the method for starting up a gas isolation section of a benzine pyrolysis unit, which includes starting the demethanizer in a steaming mode, starting the ethane and ethylene and propane and propylene towers, filling the ethylene cooling system with a hydrocarbon fraction, starting the ethylene cooling system, switching to a mode of the demethanization unit, starting the subassemblies for hydration and isolation of the ethylene and propylene fractions, in order to reduce the length of the start up period and to reduce the ejection of gas to the burner, after starting the demethanizer in the steaming mode, starting the ethane and ethylene and propane and propylene towers, the ethane and ethylene and propane and propylene fractions are mixed before the hydration subassemblies with an H2 bearing gas for catalytic reforming and then the other units and subassemblies are started.

  9. Differential hexosamine biosynthetic pathway gene expression with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Megan Coomer

    2014-01-01

    Full Text Available The hexosamine biosynthetic pathway (HBP culminates in the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc onto serine/threonine residues of target proteins. The HBP is regulated by several modulators, i.e. O-linked β-N-acetylglucosaminyl transferase (OGT and β-N-acetylglucosaminidase (OGA catalyze the addition and removal of O-GlcNAc moieties, respectively; while flux is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFPT, transcribed by two genes, GFPT1 and GFPT2. Since increased HBP flux is glucose-responsive and linked to insulin resistance/type 2 diabetes onset, we hypothesized that diabetic individuals exhibit differential expression of HBP regulatory genes. Volunteers (n = 60; n = 20 Mixed Ancestry, n = 40 Caucasian were recruited from Stellenbosch and Paarl (Western Cape, South Africa and classified as control, pre- or diabetic according to fasting plasma glucose and HbA1c levels, respectively. RNA was purified from leukocytes isolated from collected blood samples and OGT, OGA, GFPT1 and GFPT2 expressions determined by quantitative real-time PCR. The data reveal lower OGA expression in diabetic individuals (P < 0.01, while pre- and diabetic subjects displayed attenuated OGT expression vs. controls (P < 0.01 and P < 0.001, respectively. Moreover, GFPT2 expression decreased in pre- and diabetic Caucasians vs. controls (P < 0.05 and P < 0.01, respectively. We also found ethnic differences, i.e. Mixed Ancestry individuals exhibited a 2.4-fold increase in GFPT2 expression vs. Caucasians, despite diagnosis (P < 0.01. Gene expression of HBP regulators differs between diabetic and non-diabetic individuals, together with distinct ethnic-specific gene profiles. Thus differential HBP gene regulation may offer diagnostic utility and provide candidate susceptibility genes for different ethnic groupings.

  10. The preliminary research for biosynthetic engineering by radiation fusion technology

    Energy Technology Data Exchange (ETDEWEB)

    Roh, Chang Hyun; Jung, U Hee; Park, Hae Ran [KAERI, Daejeon (Korea, Republic of)

    2012-01-15

    The purpose of this project is to elucidate the solution to the production of bioactive substance using biotransformation process from core technology of biosynthetic engineering by radiation fusion technology. And, this strategy will provide core technology for development of drugs as new concept and category. Research scopes and contents of project include 1) The development of mutant for biosynthetic engineering by radiation fusion technology 2) The development of host for biosynthetic engineering by radiation fusion technology 3) The preliminary study for biosynthetic engineering of isoflavone by radiation fusion technology. The results are as follows. Isoflavone compounds(daidzein, hydroxylated isoflavone) were analyzed by GC-MS. The study of radiation doses and p-NCA high-throughput screening for mutant development were elucidated. And, it was carried out the study of radiation doses for host development. Furthermore, the study of redox partner and construction of recombinant strain for region-specific hydroxylation(P450, redox partner). In addition, the biological effect of 6,7,4'-trihydroxyisoflavone as an anti-obesity agent was elucidated in this study.

  11. Minimum Information about a Biosynthetic Gene cluster : commentary

    NARCIS (Netherlands)

    Medema, Marnix H; Kottmann, Renzo; Yilmaz, Pelin; Cummings, Matthew; Biggins, John B; Blin, Kai; de Bruijn, Irene; Chooi, Yit Heng; Claesen, Jan; Coates, R Cameron; Cruz-Morales, Pablo; Duddela, Srikanth; Dusterhus, Stephanie; Edwards, Daniel J; Fewer, David P; Garg, Neha; Geiger, Christoph; Gomez-Escribano, Juan Pablo; Greule, Anja; Hadjithomas, Michalis; Haines, Anthony S; Helfrich, Eric J N; Hillwig, Matthew L; Ishida, Keishi; Jones, Adam C; Jones, Carla S; Jungmann, Katrin; Kegler, Carsten; Kim, Hyun Uk; Kotter, Peter; Krug, Daniel; Masschelein, Joleen; Melnik, Alexey V; Mantovani, Simone M; Monroe, Emily A; Moore, Marcus; Moss, Nathan; Nutzmann, Hans-Wilhelm; Pan, Guohui; Pati, Amrita; Petras, Daniel; Reen, F Jerry; Rosconi, Federico; Rui, Zhe; Tian, Zhenhua; Tobias, Nicholas J; Tsunematsu, Yuta; Wiemann, Philipp; Wyckoff, Elizabeth; Yan, Xiaohui; Yim, Grace; Yu, Fengan; Xie, Yunchang; Aigle, Bertrand; Apel, Alexander K; Balibar, Carl J; Balskus, Emily P; Barona-Gomez, Francisco; Bechthold, Andreas; Bode, Helge B; Borriss, Rainer; Brady, Sean F; Brakhage, Axel A; Caffrey, Patrick; Cheng, Yi-Qiang; Clardy, Jon; Cox, Russell J; De Mot, Rene; Donadio, Stefano; Donia, Mohamed S; van der Donk, Wilfred A; Dorrestein, Pieter C; Doyle, Sean; Driessen, Arnold J M; Ehling-Schulz, Monika; Entian, Karl-Dieter; Fischbach, Michael A; Gerwick, Lena; Gerwick, William H; Gross, Harald; Gust, Bertolt; Hertweck, Christian; Hofte, Monica; Jensen, Susan E; Ju, Jianhua; Katz, Leonard; Kaysser, Leonard; Klassen, Jonathan L; Keller, Nancy P; Kormanec, Jan; Kuipers, Oscar P; Kuzuyama, Tomohisa; Kyrpides, Nikos C; Kwon, Hyung-Jin; Lautru, Sylvie; Lavigne, Rob; Lee, Chia Y; Linquan, Bai; Liu, Xinyu; Liu, Wen; Luzhetskyy, Andriy; Mahmud, Taifo; Mast, Yvonne; Mendez, Carmen; Metsa-Ketela, Mikko; Micklefield, Jason; Mitchell, Douglas A; Moore, Bradley S; Moreira, Leonilde M; Muller, Rolf; Neilan, Brett A; Nett, Markus; Nielsen, Jens; O'Gara, Fergal; Oikawa, Hideaki; Osbourn, Anne; Osburne, Marcia S; Ostash, Bohdan; Payne, Shelley M; Pernodet, Jean-Luc; Petricek, Miroslav; Piel, Jorn; Ploux, Olivier; Raaijmakers, Jos M; Salas, Jose A; Schmitt, Esther K; Scott, Barry; Seipke, Ryan F; Shen, Ben; Sherman, David H; Sivonen, Kaarina; Smanski, Michael J; Sosio, Margherita; Stegmann, Evi; Sussmuth, Roderich D; Tahlan, Kapil; Thomas, Christopher M; Tang, Yi; Truman, Andrew W; Viaud, Muriel; Walton, Jonathan D; Walsh, Christopher T; Weber, Tilmann; van Wezel, Gilles P; Wilkinson, Barrie; Willey, Joanne M; Wohlleben, Wolfgang; Wright, Gerard D; Ziemert, Nadine; Zhang, Changsheng; Zotchev, Sergey B; Breitling, Rainer; Takano, Eriko; Glockner, Frank Oliver

    A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit.

  12. Expression profile of genes coding for carotenoid biosynthetic ...

    Indian Academy of Sciences (India)

    Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits. Shuchi Smita, Ravi Rajwanshi, Sangram Keshari Lenka, Amit Katiyar, Viswanathan Chinnusamy and. Kailash Chander Bansal. J. Genet. 92, 363–368. Table 1.

  13. Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings.

    Science.gov (United States)

    Kubasek, WL; Shirley, BW; McKillop, A; Goodman, HM; Briggs, W; Ausubel, FM

    1992-01-01

    Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor. PMID:12297632

  14. Phene Plate (PhP) biochemical fingerprinting. A screening method for epidemiological typing of enterococcal isolates.

    Science.gov (United States)

    Saeedi, B; Tärnberg, M; Gill, H; Hällgren, A; Jonasson, J; Nilsson, L E; Isaksson, B; Kühn, I; Hanberger, H

    2005-09-01

    Pulsed-field gel electrophoresis (PFGE) is currently considered the gold standard for genotyping of enterococci. However, PFGE is both expensive and time-consuming. The purpose of this study was to investigate whether the PhP system can be used as a reliable clinical screening method for detection of genetically related isolates of enterococci. If so, it should be possible to minimize the number of isolates subjected to PFGE typing, which would save time and money. Ninety-nine clinical enterococcal isolates were analysed by PhP (similarity levels 0.90-0.975) and PFGE (similarity levels PhP also belong to the same cluster according to PFGE, i.e. p(A(PFGE)=B(PFGE) * A(PhP)=B(PhP)), and the probability that a pair of isolates of different types according to PhP also belong to different clusters according to PFGE, i.e. p(A(PFGE) not equalB(PFGE) * A(PhP) not equalB(PhP)), was relatively high for E. faecalis (0.86 and 0.96, respectively), but was lower for E. faecium (0.51 and 0.77, respectively). The concordance which shows the probability that PhP and PFGE agree on match or mismatch was 86%-93% for E. faecalis and 54%-66% for E. faecium, which indicates that the PhP method may be useful for epidemiological typing of E. faecalis in the current settings but not for E. faecium.

  15. Comparison of Methods for Isolating High Quality DNA and RNA from an Oleaginous Fungus Cunninghamella bainieri Strain 2a1

    OpenAIRE

    Noor Adila, A. K.; Farah Diba, A. B.; Zamri, Z.; Wan Mohtar, W. Y.; Aidil, A. H.; Mahadi, N. M.; Murad, A. M. A.

    2007-01-01

    A number of protocols have been reported for efficient fungal DNA and RNA isolation. However, many of these methods are often designed for certain groups or morphological forms of fungi and, in some cases, are species dependent. In this report, we compared four published protocols for DNA isolation from a locally isolated oleaginous fungus, Cunninghamella bainieri strain 2a1. These protocols either involved the use of polyvinyl pyrrolidone (PVP), hexacetyltrimethylammonium bromide (CTAB) or w...

  16. Elucidating the biosynthetic and regulatory mechanisms of flavonoid-derived bioactive components in Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun eHuang

    2015-09-01

    Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. In Epimedium, flavonoids have been demonstrated to be the main bioactive components (BCs. However, the molecular biosynthetic and regulatory mechanisms of flavonoid-derived BCs remain obscure. In this study, we isolated twelve structural genes and two putative transcription factors (TFs in the flavonoid pathway. Phytochemical analysis showed that the total content of four representative BCs (epimedin A, B, C and icariin decreased slightly or dramatically in two lines of E. sagittatum during leaf development. Transcriptional analysis revealed that two R2R3-MYB TFs (EsMYBA1 and EsMYBF1, together with a bHLH TF (EsGL3 and WD40 protein (EsTTG1, were supposed to coordinately regulate the anthocyanin and flavonol-derived BCs biosynthesis in leaves. Overexpression of EsFLS (flavonol synthase in tobacco resulted in increased flavonols content and decreased anthocyanins content in flowers. Moreover, EsMYB12 negatively correlated with the accumulation of the four BCs, and might act as a transcriptional repressor in the flavonoid pathway. Therefore, the anthocyanin pathway may coordinate with the flavonol-derived BCs pathway in Epimedium leaves. A better understanding of the flavonoid biosynthetic and regulatory mechanisms in E. sagittatum will facilitate functional characterization, metabolic engineering and molecular breeding studies of Epimedium species.

  17. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  18. Treadmill exercise does not change gene expression of adrenal catecholamine biosynthetic enzymes in chronically stressed rats

    Directory of Open Access Journals (Sweden)

    LJUBICA GAVRILOVIC

    2013-09-01

    Full Text Available ABSTRACT Chronic isolation of adult animals represents a form of psychological stress that produces sympatho-adrenomedullar activation. Exercise training acts as an important modulator of sympatho-adrenomedullary system. This study aimed to investigate physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase and phenylethanolamine N-methyltransferase and cyclic adenosine monophosphate response element-binding (CREB in the adrenal medulla, concentrations of catecholamines and corticosterone (CORT in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill running for 12 weeks. Also, we examined how additional acute immobilization stress changes the mentioned parameters. Treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes and it decreased the level of CREB mRNA in the adrenal medulla of chronically psychosocially stressed adult rats. The potentially negative physiological adaptations after treadmill running were recorded as increased concentrations of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy of chronically psychosocially stressed rats. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. Treadmill exercise does not change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats.

  19. Expression of phenazine biosynthetic genes during the arbuscular mycorrhizal symbiosis of Glomus intraradices

    Directory of Open Access Journals (Sweden)

    Dionicia Gloria León-Martínez

    2012-06-01

    Full Text Available To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010. Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

  20. Isolation and Identification of Volatile Components in Tempe by Simultaneous Distillation-Extraction Method by Modified Extraction Method

    Directory of Open Access Journals (Sweden)

    Syahrial Syahrial

    2010-06-01

    Full Text Available An isolation and identification of volatile components in temps for 2, 5 and 8 days fermentation by simultaneous distillation-extraction method was carried out. Simultaneous distillation-extraction apparatus was modified by Muchalal from the basic Likens-Nickerson's design. Steam distillation and benzena as an extraction solvent was used in this system. The isolation was continuously carried out for 3 hours which maximum water temperature In the Liebig condenser was 8 °C. The extract was concentrated by freeze concentration method, and the volatile components were analyzed and identified by combined gas chromatography-mass spectrophotometry (GC-MS. The Muchalal's simultaneous distillation extraction apparatus have some disadvantage in cold finger condenser, and it's extractor did not have condenser. At least 47, 13 and 5 volatile components were found in 2, 5 and 8 days fermentation, respectively. The volatile components in the 2 days fermentation were nonalal, ɑ-pinene, 2,4-decadienal, 5-phenyldecane, 5-phenylundecane, 4-phenylundecane, 5-phenyldodecane, 4-phenyldodecane, 3-phenyldodecane, 2-phenyldodecane, 5-phenyltridecane, and caryophyllene; in the 5 days fermentation were nonalal, caryophyllene, 4-phenylundecane, 5-phenyldodecane, 4-phenyldodecane, 3-phenyldodecane, 2-phenyldodecane; and in the 8 days fermentation were ethenyl butanoic, 2-methy1-3-(methylethenylciclohexyl etanoic and 3,7-dimethyl-5-octenyl etanoic.

  1. Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high-performance countercurrent chromatography.

    Science.gov (United States)

    Rho, Taewoong; Jung, Mila; Lee, Min Won; Chin, Young-Won; Yoon, Kee Dong

    2016-12-01

    Efficient high-performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n-Butanol-soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid (1), swertiamarin (2) and gentiopicroside (3), and a chromene derivative, macrophylloside D (4) which were successfully isolated by flow rate gradient (1.5 mL/min in 0-60 min, 5.0 mL/min in 60-120 min), and consecutive flow rate gradient HPCCC using n-butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1-4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high-performance countercurrent chromatography, and consecutive flow rate gradient high-performance countercurrent chromatography gave 1, 2, 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2-methoxyanofinic acid, was successfully separated by n-hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow-rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by 1 H, 13 C NMR and ESI-Q-TOF-MS spectroscopic data which were compared with previously reported values. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

    Science.gov (United States)

    Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

    2014-01-01

    The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

  3. Methods for Confirming the Gram Reaction of Gram-variable Bacillus Species Isolated from Tobacco

    Directory of Open Access Journals (Sweden)

    Morin A

    2014-12-01

    Full Text Available Bacillus is a predominant genus of bacteria isolated from tobacco. The Gram stain is the most commonly used and most important of all diagnostic staining techniques in microbiology. In order to help confirm the Gram positivity of Bacillus isolates from tobacco, three methods using the chemical differences of the cell wall and membrane of Gram-positive and Gram-negative bacteria were investigated: the KOH (potassium hydroxide, the LANA (L-alanine-4-nitroanilide, and the vancomycin susceptibility tests. When colonies of Gram-negative bacteria are treated with 3% KOH solution, a slimy suspension is produced, probably due to destruction of the cell wall and liberation of deoxyribonucleic acid (DNA. Gram-positive cell walls resist KOH treatment. The LANA test reveals the presence of a cell wall aminopeptidase that hydrolyzes the L-alanine-4-nitroanilide in Gram-negative bacteria. This enzyme is absent in Gram-positive bacteria. Vancomycin is a glycopeptide antibiotic inhibiting the cell wall peptido-glycan synthesis of Gram-positive microorganisms. Absence of lysis with KOH, absence of hydrolysis of LANA, and susceptibility to vancomycin were used with the Gram reaction to confirm the Gram positivity of various Bacillus species isolated from tobacco. B. laevolacticus excepted, all Bacillus species tested showed negative reactions to KOH and LANA tests, and all species were susceptible to vancomycin (5 and 30 µg.

  4. In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

    Directory of Open Access Journals (Sweden)

    Santanu Panda

    Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

  5. Effects of Phytophthora cinnamomi isolate, inoculum delivery method, flood, and drought on vigor, disease severity and mortality of blueberry plants

    Science.gov (United States)

    Four studies evaluated the effect of Phytophthora cinnamomi isolates, inoculum delivery methods, and flood and drought conditions on vigor, disease severity scores, and survival of blueberry plants grown in pots in the greenhouse. Phytophthora cinnamomi isolates were obtained from blueberry plants ...

  6. New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

    Directory of Open Access Journals (Sweden)

    Karen Nava-Castro

    2011-01-01

    Full Text Available In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

  7. Inverse PCR-based method for isolating novel SINEs from genome.

    Science.gov (United States)

    Han, Yawei; Chen, Liping; Guan, Lihong; He, Shunping

    2014-04-01

    Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

  8. Automatic speech recognition (zero crossing method). Automatic recognition of isolated vowels

    International Nuclear Information System (INIS)

    Dupeyrat, Benoit

    1975-01-01

    This note describes a recognition method of isolated vowels, using a preprocessing of the vocal signal. The processing extracts the extrema of the vocal signal and the interval time separating them (Zero crossing distances of the first derivative of the signal). The recognition of vowels uses normalized histograms of the values of these intervals. The program determines a distance between the histogram of the sound to be recognized and histograms models built during a learning phase. The results processed on real time by a minicomputer, are relatively independent of the speaker, the fundamental frequency being not allowed to vary too much (i.e. speakers of the same sex). (author) [fr

  9. Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities.

    Science.gov (United States)

    Cheng, Jiujun; Nordeste, Ricardo; Trainer, Maria A; Charles, Trevor C

    2017-01-01

    Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates the functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

  10. Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities.

    Science.gov (United States)

    Nordeste, Ricardo F; Trainer, Maria A; Charles, Trevor C

    2010-01-01

    Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

  11. Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Horn, Thomas; Nielsen, Ole H

    2003-01-01

    and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different......Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy...

  12. Comparative Analysis of the Genomic DNA Isolation Methods on Inula sp. (Asteraceae

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    Emre SEVİNDİK

    2016-12-01

    Full Text Available Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280 that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method.

  13. Diversity of Culturable Thermophilic Actinobacteria in Hot Springs in Tengchong, China and Studies of their Biosynthetic Gene Profiles.

    Science.gov (United States)

    Liu, Lan; Salam, Nimaichand; Jiao, Jian-Yu; Jiang, Hong-Chen; Zhou, En-Min; Yin, Yi-Rui; Ming, Hong; Li, Wen-Jun

    2016-07-01

    The class Actinobacteria has been a goldmine for the discovery of antibiotics and has attracted interest from both academics and industries. However, an absence of novel approaches during the last few decades has limited the discovery of new microbial natural products useful for industries. Scientists are now focusing on the ecological aspects of diverse environments including unexplored or underexplored habitats and extreme environments in the search for new metabolites. This paper reports on the diversity of culturable actinobacteria associated with hot springs located in Tengchong County, Yunnan Province, southwestern China. A total of 58 thermophilic actinobacterial strains were isolated from the samples collected from ten hot springs distributed over three geothermal fields (e.g., Hehua, Rehai, and Ruidian). Phylogenetic positions and their biosynthetic profiles were analyzed by sequencing 16S rRNA gene and three biosynthetic gene clusters (KS domain of PKS-I, KSα domain of PKS-II and A domain of NRPS). On the basis of 16S rRNA gene phylogenetic analysis, the 58 strains were affiliated with 12 actinobacterial genera: Actinomadura Micromonospora, Microbispora, Micrococcus, Nocardiopsis, Nonomuraea, Promicromonospora, Pseudonocardia, Streptomyces, Thermoactinospora, Thermocatellispora, and Verrucosispora, of which the two novel genera Thermoactinospora and Thermocatellisopora were recently described from among these strains. Considering the biosynthetic potential of these actinobacterial strains, 22 were positive for PCR amplification of at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, and NRPS). These actinobacteria were further subjected to antimicrobial assay against five opportunistic human pathogens (Acinetobacter baumannii, Escherichia coli, Micrococcus luteus, Staphylococcus aureus and Streptococcus faecalis). All of the 22 strains that were positive for PCR amplification of at least one of the biosynthetic gene domains exhibited

  14. Didemnin Biosynthetic Gene Cluster In Tistrella Mobilis

    KAUST Repository

    Qian, Pei-Yuan; Xu, Ying Sharon; Lai, Pok-Yui

    2014-01-01

    A novel Tistrella mobilis strain having Accession Deposit Number NRRL B-50531 is provided. A method of producing a didemnin precursor, didemnin or didemnin derivative by using the Tistrella mobilis strain, and the therapeutic composition comprising

  15. Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Samira Mohammadi

    2017-01-01

    Full Text Available Background: Nontuberculous mycobacteria (NTM are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. Materials and Methods: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR amplification of the heat-shock protein 65 gene with serially diluted DNA samples. Results: The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. Conclusions: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

  16. Use of (/sup 75/Se)selenomethionine in immunoglobulin biosynthetic studies

    Energy Technology Data Exchange (ETDEWEB)

    Gutman, G A; Warner, N L; Harris, A W; Bowles, A [Walter and Elisa Hall Institute of Medical Research, Victoria (Australia). Genetics Unit; Royal Melbourne Hospital, Victoria (Australia))

    1978-05-01

    The gamma-emitting amino acid analog, (/sup 75/Se) selenomethionine, has been used as a biosynthetic label for immunoglobulins secreted by plasmacytomas in tissue culture. The secreted products are structurally intact with respect to their antibody combining sites and their class and allotype antigenic specificities. A component of (/sup 75/Se) selenomethionine preparations was found to bind to fetal calf serum proteins, in a manner releasable by mercaptoethanol, but not by sodium dodecyl sulfate and urea. Methods for circumventing the problems caused by this binding are described.

  17. A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels.

    Science.gov (United States)

    Brzica, Hrvoje; Abdullahi, Wazir; Reilly, Bianca G; Ronaldson, Patrick T

    2018-05-07

    The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting from these stimuli can greatly modulate drug delivery to the brain and, by extension, cause considerable challenges in the treatment of central nervous system (CNS) diseases. Many BBB changes that affect pharmacotherapy, involve proteins that are localized and expressed at the level of endothelial cells. Indeed, such knowledge on BBB physiology in health and disease has sparked considerable interest in the study of these membrane proteins. From a basic science research standpoint, this implies a requirement for a simple but robust and reproducible method for isolation of microvessels from brain tissue harvested from experimental animals. In order to prepare membrane samples from freshly isolated microvessels, it is essential that sample preparations be enriched in endothelial cells but limited in the presence of other cell types of the neurovascular unit (i.e., astrocytes, microglia, neurons, pericytes). An added benefit is the ability to prepare samples from individual animals in order to capture the true variability of protein expression in an experimental population. In this manuscript, details regarding a method that is utilized for isolation of rat brain microvessels and preparation of membrane samples are provided. Microvessel enrichment, from samples derived, is achieved by using four centrifugation steps where dextran is included in the sample buffer. This protocol can easily be adapted by other laboratories for their own specific applications. Samples generated from this protocol have been shown to yield robust experimental data from protein analysis experiments that can greatly aid the understanding of BBB responses to physiological, pathophysiological, and pharmacological stimuli.

  18. PRODUCTION OF EMBBRYONIC STEM CELLS FROM INNER CELL MASS OF BLASTOCYST ISOLATED BY ENZYMATIC AND IMMUNOSURGERY METHODS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2008-03-01

    Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

  19. Oral rinse as a potential method to culture Candida isolate from AIDS patients

    Directory of Open Access Journals (Sweden)

    Desiana Radithia

    2011-12-01

    Full Text Available Background: Candida isolate is easily sampled from oral cavity by swabbing directly on the candidiasis lesion, to be smeared onto slides for direct examination or cultured in a growth medium. This method is by far the gold standard for defining candidiasis diagnosis. However it is difficult to apply on sensitive patients and almost impossible on patients showing no clinical appearance of oral candidiasis. AIDS patients are very prone to candida infection and have a tendency of repetitive infection involving mixed species. As many candida species show different susceptibility to anti-fungal agents, it is necessary to identify the species causing the infection in the management of oral candidiasis. Oral rinse is a suggested method to obtain candida isolate to be cultured for further analysis such as species identification. This method is simple and less risky on infection transmission as less tools are required in the procedure. Purpose: This study aimed to assess the application of oral rinse as an alternative method to culture Candida isolate from AIDS patients. Methods: A cross-sectional observative study was conducted in HIV/AIDS in-Patient Facility of Intermediate Care Unit for Infection Disease, Dr. Soetomo Hospital Surabaya. Fourteen stadium 4 AIDS patients matching criteria were swabbed on 1/3-posterior of the tongue, and then given 10 ml phosphate buffer saline to rinse vigorously for 15 seconds. Both specimens were cultured on Sabouraud’s dextrose agar and colony growth was observed. Results: Candida colonies were able to grow from all 14 isolates (100% by both methods. Qualitatively, cultures from oral rinse specimens were more populated than cultures from swab specimens. Conclusion: Oral rinse is an applicable technique to obtain Candida species isolate. This technique is safe, easy, non-invasive, and needs less tools therefore less risky for HIV transmission.Latar belakang: Isolat Candida mudah diambil dengan cara mengusap lesi

  20. A method to determine insulin responsiveness in synaptosomes isolated from frozen brain tissue.

    Science.gov (United States)

    Franklin, Whitney; Taglialatela, Giulio

    2016-03-01

    Studying the insulin signaling response at the synapse is an important approach to understand molecular mechanisms involved in disease-related neurodegenerative processes. We developed a method for studying the insulin responsiveness at the synaptic level by isolating functional synaptosomes from fresh or frozen tissue and exposing them to insulin in the presence of ATP (a critical step) to detect insulin receptor (IR) activation. We performed an ATP dose-response curve, insulin dose-response curve, and insulin response time course to optimize this method. We also demonstrated that our protocol reflects the degree of insulin responsiveness in vivo by using an animal model of known insulin resistance, AtENPP1-Tg mice. This method is advantageous over other methods detecting IR in total brain homogenates due to the ability to detect IR response without confounding contributions from other cell areas and cell types also expressing IR. Furthermore, ex vivo insulin stimulation can be compared to baseline synaptosomes obtained from the same animal which improves reliability and statistical power while decreasing the number of animals required to perform individual experiments. We have developed a reliable, efficient method to measure insulin-driven ex vivo phosphorylation of the synaptosomal insulin receptor that can reliably reflect the pre-existing insulin responsiveness status in the CNS of the animal. To the best of our knowledge, this is the first evidence of stimulation of isolated synaptosomes with insulin and a promising new technique to study the synaptic CNS insulin responsiveness under physiological or disease conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Isolation of uv-sensitive variants of human FL cells by a viral suicide method

    International Nuclear Information System (INIS)

    Shiomi, T.; Sato, K.

    1979-01-01

    A new method (viral suicide method) for the isolation of uv-sensitive mutants is described. Colonies of mutagenized human FL cells were infected with uv-irradiated Herpes simplex viruses and surviving ones which seemed to be deficient in host cell reactivation (HCR) were examined for their uv sensitivity. Nineteen of 238 clones examined were sensitive to uv irradiation at the time of the isolation. After recloning, four of these clones have been studied and two (UVS-1 and UVS-2) of them are stable in their uv sensitivity for 4 months in culture. uv sensitivity of UVS-1, UVS-2, and the parental FL cells are as follows: the extrapolation numbers (n) are 2.2, 2.1, and 1.8 and mean lethal doses (DO) are 2.9, 3.7, and 7.8 J/m 2 for UVS-1, UVS-2, and the parental FL cells, respectively. They are no more sensitive than FL cells to x-irradiation. The ability of HCR in UVS-2 cells is apparently lower than that in FL cells, whereas UVS-1 cells are the same as FL cells in the ability

  2. Evaluation of three methods for DNA fingerprinting of Corynebacterium pseudotuberculosis strains isolated from goats in Poland.

    Science.gov (United States)

    Stefańska, Ilona; Rzewuska, Magdalena; Binek, Marian

    2008-01-01

    Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.

  3. Identification of Mucorales isolates from soil using morphological and molecular methods

    Directory of Open Access Journals (Sweden)

    Ardeshir Ziaee

    2016-03-01

    Full Text Available Background and Purpose: Soil is the main habitat of saprophytic and pathogenic fungi. Mucormycetes are one of the most parts of soil fungi and certain members are among opportunistic fungi and can cause systemic fungal infections in immunocompromised patients. The majority of human and animal infections are caused by members of the genera Rhizopus, Mucor, Rhizomucor, Lichtheimia (Absidia, Cunninghamella and Mortierella. The objective of this research was to isolate and identify the main genera of Zygomycetes, using molecular assay and morphological features. Materials and Methods: A total of 340 soil samples were collected from different sites of seven public parks and 14 municipality districts in Isfahan. All samples were cultured on appropriate media and incubated at 27° C for 2 to 4 days, and then examined daily for visible fungal growth. PCR-RFLP method and macroscopic, microscopic and physiological characteristics were applied to identify fungal colonies. Results: Four hundred pure colonies belonging to six genera of Zygomycetes including Lichtheimia, Rhizopus, Rhizomucor, Mucor, Cunninghamella and Mortierella were identified. The genus Rhizopus (35.5% was the most frequent isolate, followed by Mucor (32.25% and Rhizomucor (27.5%. Conclusion: These finding may help us to understand about the importance of opportunistic fungi in public areas and the risk of exposure with immunocompromised persons.

  4. Cell fusion in tumor progression: the isolation of cell fusion products by physical methods

    Directory of Open Access Journals (Sweden)

    Vincitorio Massimo

    2011-09-01

    Full Text Available Abstract Background Cell fusion induced by polyethylene glycol (PEG is an efficient but poorly controlled procedure for obtaining somatic cell hybrids used in gene mapping, monoclonal antibody production, and tumour immunotherapy. Genetic selection techniques and fluorescent cell sorting are usually employed to isolate cell fusion products, but both procedures have several drawbacks. Results Here we describe a simple improvement in PEG-mediated cell fusion that was obtained by modifying the standard single-step procedure. We found that the use of two PEG undertreatments obtains a better yield of cell fusion products than the standard method, and most of these products are bi- or trinucleated polykaryocytes. Fusion rate was quantified using fluorescent cell staining microscopy. We used this improved cell fusion and cell isolation method to compare giant cells obtained in vitro and giant cells obtained in vivo from patients with Hodgkin's disease and erythroleukemia. Conclusions In the present study we show how to improve PEG-mediated cell fusion and that cell separation by velocity sedimentation offers a simple alternative for the efficient purification of cell fusion products and to investigate giant cell formation in tumor development.

  5. Optimal DNA Isolation Method for Detection of Nontuberculous Mycobacteria by Polymerase Chain Reaction.

    Science.gov (United States)

    Mohammadi, Samira; Esfahani, Bahram Nasr; Moghim, Sharareh; Mirhendi, Hossein; Zaniani, Fatemeh Riyahi; Safaei, Hajieh Ghasemian; Fazeli, Hossein; Salehi, Mahshid

    2017-01-01

    Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA ® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. The CTAB method showed more positive results at 1:10-1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.

  6. Didemnin Biosynthetic Gene Cluster In Tistrella Mobilis

    KAUST Repository

    Qian, Pei-Yuan

    2014-10-02

    A novel Tistrella mobilis strain having Accession Deposit Number NRRL B-50531 is provided. A method of producing a didemnin precursor, didemnin or didemnin derivative by using the Tistrella mobilis strain, and the therapeutic composition comprising at least one didemnin or didemnin derivative produced from the strain or modified strain thereof are also provided.

  7. Molecular characterization of tocopherol biosynthetic genes in sweetpotato that respond to stress and activate the tocopherol production in tobacco.

    Science.gov (United States)

    Ji, Chang Yoon; Kim, Yun-Hee; Kim, Ho Soo; Ke, Qingbo; Kim, Gun-Woo; Park, Sung-Chul; Lee, Haeng-Soon; Jeong, Jae Cheol; Kwak, Sang-Soo

    2016-09-01

    Tocopherol (vitamin E) is a chloroplast lipid that is presumed to be involved in the plant response to oxidative stress. In this study, we isolated and characterized five tocopherol biosynthetic genes from sweetpotato (Ipomoea batatas [L.] Lam) plants, including genes encoding 4-hydroxyphenylpyruvate dioxygenase (IbHPPD), homogentisate phytyltransferase (IbHPT), 2-methyl-6-phytylbenzoquinol methyltransferase (IbMPBQ MT), tocopherol cyclase (IbTC) and γ-tocopherol methyltransferase (IbTMT). Fluorescence microscope analysis indicated that four proteins localized into the chloroplast, whereas IbHPPD observed in the nuclear. Quantitative RT-PCR analysis revealed that the expression patterns of the five tocopherol biosynthetic genes varied in different plant tissues and under different stress conditions. All five genes were highly expressed in leaf tissues, whereas IbHPPD and IbHPT were highly expressed in the thick roots. The expression patterns of these five genes significantly differed in response to PEG, NaCl and H2O2-mediated oxidative stress. IbHPPD was strongly induced following PEG and H2O2 treatment and IbHPT was strongly induced following PEG treatment, whereas IbMPBQ MT and IbTC were highly expressed following NaCl treatment. Upon infection of the bacterial pathogen Pectobacterium chrysanthemi, the expression of IbHPPD increased sharply in sweetpotato leaves, whereas the expression of the other genes was reduced or unchanged. Additionally, transient expression of the five tocopherol biosynthetic genes in tobacco (Nicotiana bentamiana) leaves resulted in increased transcript levels of the transgenes expressions and tocopherol production. Therefore, our results suggested that the five tocopherol biosynthetic genes of sweetpotato play roles in the stress defense response as transcriptional regulators of the tocopherol production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. Phenylpropanoids accumulation in eggplant fruit: characterization of biosynthetic genes and regulation by a MYB transcription factor

    Directory of Open Access Journals (Sweden)

    Teresa eDocimo

    2016-01-01

    Full Text Available Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena fruits. Chlorogenic acid (CGA accounts for 70 to 90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena.Higher contents of CGA, Delphinidin 3-rutinoside and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group 6 MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties.In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation.Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9 resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of

  9. iPBS: a universal method for DNA fingerprinting and retrotransposon isolation.

    Science.gov (United States)

    Kalendar, Ruslan; Antonius, Kristiina; Smýkal, Petr; Schulman, Alan H

    2010-11-01

    Molecular markers are essential in plant and animal breeding and biodiversity applications, in human forensics, and for map-based cloning of genes. The long terminal repeat (LTR) retrotransposons are well suited as molecular markers. As dispersed and ubiquitous transposable elements, their "copy and paste" life cycle of replicative transposition leads to new genome insertions without excision of the original element. Both the overall structure of retrotransposons and the domains responsible for the various phases of their replication are highly conserved in all eukaryotes. Nevertheless, up to a year has been required to develop a retrotransposon marker system in a new species, involving cloning and sequencing steps as well as the development of custom primers. Here, we describe a novel PCR-based method useful both as a marker system in its own right and for the rapid isolation of retrotransposon termini and full-length elements, making it ideal for "orphan crops" and other species with underdeveloped marker systems. The method, iPBS amplification, is based on the virtually universal presence of a tRNA complement as a reverse transcriptase primer binding site (PBS) in LTR retrotransposons. The method differs from earlier retrotransposon isolation methods because it is applicable not only to endogenous retroviruses and retroviruses, but also to both Gypsy and Copia LTR retrotransposons, as well as to non-autonomous LARD and TRIM elements, throughout the plant kingdom and to animals. Furthermore, the inter-PBS amplification technique as such has proved to be a powerful DNA fingerprinting technology without the need for prior sequence knowledge.

  10. Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones.

    Science.gov (United States)

    Ibdah, Mwafaq; Martens, Stefan; Gang, David R

    2018-03-14

    Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.

  11. [Advance in flavonoids biosynthetic pathway and synthetic biology].

    Science.gov (United States)

    Zou, Li-Qiu; Wang, Cai-Xia; Kuang, Xue-Jun; Li, Ying; Sun, Chao

    2016-11-01

    Flavonoids are the valuable components in medicinal plants, which possess a variety of pharmacological activities, including anti-tumor, antioxidant and anti-inflammatory activities. There is an unambiguous understanding about flavonoids biosynthetic pathway, that is,2S-flavanones including naringenin and pinocembrin are the skeleton of other flavonoids and they can transform to other flavonoids through branched metabolic pathway. Elucidation of the flavonoids biosynthetic pathway lays a solid foundation for their synthetic biology. A few flavonoids have been produced in Escherichia coli or yeast with synthetic biological technologies, such as naringenin, pinocembrin and fisetin. Synthetic biology will provide a new way to get valuable flavonoids and promote the research and development of flavonoid drugs and health products, making flavonoids play more important roles in human diet and health. Copyright© by the Chinese Pharmaceutical Association.

  12. Functional Characterization of a Novel R2R3-MYB Transcription Factor Modulating the Flavonoid Biosynthetic Pathway from Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2017-07-01

    Full Text Available Epimedium species have been widely used both as traditional Chinese medicinal plants and ornamental perennials. Both flavonols, acting as the major bioactive components (BCs and anthocyanins, predominantly contributing to the color diversity of Epimedium flowers belong to different classes of flavonoids. It is well-acknowledged that flavonoid biosynthetic pathway is predominantly regulated by R2R3-MYB transcription factor (TF as well as bHLH TF and WD40 protein at the transcriptional level. MYB TFs specifically regulating anthocyanin or flavonol biosynthetic pathway have been already isolated and functionally characterized from Epimedium sagittatum, but a R2R3-MYB TF involved in regulating both these two pathways has not been functionally characterized to date in Epimedium plants. In this study, we report the functional characterization of EsMYB9, a R2R3-MYB TF previously isolated from E. sagittatum. The previous study indicated that EsMYB9 belongs to a small subfamily of R2R3-MYB TFs containing grape VvMYB5a and VvMYB5b TFs, which regulate flavonoid biosynthetic pathway. The present studies show that overexpression of EsMYB9 in tobacco leads to increased transcript levels of flavonoid pathway genes and increased contents of anthocyanins and flavonols. Yeast two-hybrid assay indicates that the C-terminal region of EsMYB9 contributes to the autoactivation activity, and EsMYB9 interacts with EsTT8 or AtTT8 bHLH regulator. Transient reporter assay shows that EsMYB9 slightly activates the expression of EsCHS (chalcone synthase promoter in transiently transformed leaves of Nicotiana benthamiana, but the addition of AtTT8 or EsTT8 bHLH regulator strongly enhances the transcriptional activation of EsMYB9 against five promoters of the flavonoid pathway genes except EsFLS (flavonol synthase. In addition, co-transformation of EsMYB9 and EsTT8 in transiently transfected tobacco leaves strongly induces the expressions of flavonoid biosynthetic genes. The

  13. Summary of the systems prioritization method as a decision-aiding method for the waste isolation pilot plant

    International Nuclear Information System (INIS)

    Boak, D.M.; Prindle, N.H.; Lincoln, R.

    1996-12-01

    In March 1994, the U.S. Department of Energy Carlsbad Area Office (DOE/CAO) implemented a performance-based decision-aiding method to assist in programmatic prioritization within the Waste Isolation Pilot Plant (WIPP) Project with respect to applicable U.S. Environmental Protection Agency (EPA) long-term performance requirements in 40 CFR 191.13(a) (radionuclide containment requirements) and 40 CFR 268.6 (hazardous constituent concentration requirements). This method, the Systems Prioritization Method (SPM), was designed by Sandia National Laboratories (SNL) to: (1) identify programmatic options (activities) and their costs and durations; (2) analyze combinations of activities (activity sets) in terms of their predicted contribution to long-term performance of the WIPP disposal system; and (3) analyze cost, duration, and performance tradeoffs. The results of the second iteration of SPM (SPM-2) were the basis for recommendations to DOE/CAO in May 1995 for programmatic prioritization within the WIPP project. This paper presents a summary of the SPM implementation, key results, and lessons learned

  14. Summary of the systems prioritization method as a decision-aiding method for the waste isolation pilot plant

    Energy Technology Data Exchange (ETDEWEB)

    Boak, D.M.; Prindle, N.H.; Lincoln, R. [and others

    1996-12-01

    In March 1994, the U.S. Department of Energy Carlsbad Area Office (DOE/CAO) implemented a performance-based decision-aiding method to assist in programmatic prioritization within the Waste Isolation Pilot Plant (WIPP) Project with respect to applicable U.S. Environmental Protection Agency (EPA) long-term performance requirements in 40 CFR 191.13(a) (radionuclide containment requirements) and 40 CFR 268.6 (hazardous constituent concentration requirements). This method, the Systems Prioritization Method (SPM), was designed by Sandia National Laboratories (SNL) to: (1) identify programmatic options (activities) and their costs and durations; (2) analyze combinations of activities (activity sets) in terms of their predicted contribution to long-term performance of the WIPP disposal system; and (3) analyze cost, duration, and performance tradeoffs. The results of the second iteration of SPM (SPM-2) were the basis for recommendations to DOE/CAO in May 1995 for programmatic prioritization within the WIPP project. This paper presents a summary of the SPM implementation, key results, and lessons learned.

  15. Evaluating Methods for Isolating Total RNA and Predicting the Success of Sequencing Phylogenetically Diverse Plant Transcriptomes

    Science.gov (United States)

    Bruskiewich, Richard; Burris, Jason N.; Carrigan, Charlotte T.; Chase, Mark W.; Clarke, Neil D.; Covshoff, Sarah; dePamphilis, Claude W.; Edger, Patrick P.; Goh, Falicia; Graham, Sean; Greiner, Stephan; Hibberd, Julian M.; Jordon-Thaden, Ingrid; Kutchan, Toni M.; Leebens-Mack, James; Melkonian, Michael; Miles, Nicholas; Myburg, Henrietta; Patterson, Jordan; Pires, J. Chris; Ralph, Paula; Rolf, Megan; Sage, Rowan F.; Soltis, Douglas; Soltis, Pamela; Stevenson, Dennis; Stewart, C. Neal; Surek, Barbara; Thomsen, Christina J. M.; Villarreal, Juan Carlos; Wu, Xiaolei; Zhang, Yong; Deyholos, Michael K.; Wong, Gane Ka-Shu

    2012-01-01

    Next-generation sequencing plays a central role in the characterization and quantification of transcriptomes. Although numerous metrics are purported to quantify the quality of RNA, there have been no large-scale empirical evaluations of the major determinants of sequencing success. We used a combination of existing and newly developed methods to isolate total RNA from 1115 samples from 695 plant species in 324 families, which represents >900 million years of phylogenetic diversity from green algae through flowering plants, including many plants of economic importance. We then sequenced 629 of these samples on Illumina GAIIx and HiSeq platforms and performed a large comparative analysis to identify predictors of RNA quality and the diversity of putative genes (scaffolds) expressed within samples. Tissue types (e.g., leaf vs. flower) varied in RNA quality, sequencing depth and the number of scaffolds. Tissue age also influenced RNA quality but not the number of scaffolds ≥1000 bp. Overall, 36% of the variation in the number of scaffolds was explained by metrics of RNA integrity (RIN score), RNA purity (OD 260/230), sequencing platform (GAIIx vs HiSeq) and the amount of total RNA used for sequencing. However, our results show that the most commonly used measures of RNA quality (e.g., RIN) are weak predictors of the number of scaffolds because Illumina sequencing is robust to variation in RNA quality. These results provide novel insight into the methods that are most important in isolating high quality RNA for sequencing and assembling plant transcriptomes. The methods and recommendations provided here could increase the efficiency and decrease the cost of RNA sequencing for individual labs and genome centers. PMID:23185583

  16. Isolation and purification of Echinococcus granulosus antigen B from hydatid cyst fluid using three different methods

    Directory of Open Access Journals (Sweden)

    Shirazi, S.

    2016-07-01

    Full Text Available Hydatid cyst, the larval stage of cestodes Echinococcus spp., is recognized as a zoonotic infection in the world. The World Health Organization (WHO has recently classified echinococcosis in a group of neglected tropical diseases. The prevalence of Echinococcus granulosus infection is high in Iran due to the presence of various intermediate hosts in this country. Considering the rising trend of this zoonotic parasitic disease based on national epidemiological studies, diagnosis is of great significance. WHO has suggested the use of specific antigens, especially antigen B (AgB for serological diagnostic tests. In general, AgB is a polymeric lipoprotein, which disintegrates into 8.12, 16, and 20.24 kDa subunits. In the present study, we applied three different methods for AgB isolation from hydatid cyst fluid (HCF and compared their efficacy in AgB isolation. Finally, the protein concentration of this antigen was measured by Bradford assay and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE. The results showed that the application of polyethylene glycol (PEG 4000 as a thickener agent beside purification of HCF in dialysis bag and filtering and also dialysis against acetate buffer leading to the best quantity in purified antigen B.

  17. A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae).

    Science.gov (United States)

    Vazquez-Angulo, J C; Mendez-Trujillo, V; González-Mendoza, D; Morales-Trejo, A; Grimaldo-Juarez, O; Cervantes-Díaz, L

    2012-05-15

    Extraction of high-quality genomic DNA for PCR amplification from filamentous fungi is difficult because of the complex cell wall and the high concentrations of polysaccharides and other secondary metabolites that bind to or co-precipitate with nucleic acids. We developed a modified sodium dodecyl sulfate/phenol protocol, without maceration in liquid nitrogen and without a final ethanol precipitation step. The A(260/280) absorbance ratios of isolated DNA were approximately 1.7-1.9, demonstrating that the DNA fraction is pure and can be used for analysis. Additionally, the A(260/230) values were higher than 1.6, demonstrating negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. The main advantages of the method are that the mycelium is directly recovered from culture medium and it does not require the use of expensive and specialized equipment.

  18. Identification of Mucorales isolates from soil using morphological and molecular methods.

    Science.gov (United States)

    Ziaee, A; Zia, M; Bayat, M; Hashemi, J

    2016-03-01

    Soil is the main habitat of saprophytic and pathogenic fungi. Mucoromycotina constitutes a large group of soil fungi, with certain opportunistic members causing systemic infections in immunocompromised hosts. The majority of human and animal infections are caused by the members of the genera Rhizopus , Mucor , Rhizomucor , Lichtheimia ( Absidia) , Cunninghamella, and Mortierella . Accordingly, in the present study, we aimed to isolate and identify the main genera of the order Mucorales, using molecular assays and morphological features . In total, 340 soil samples were collected from seven public parks throughout the city and sidewalk gardens in 14 municipal districts in Isfahan, Iran. All the samples were cultured on the appropriate media, incubated at 27°C for 2- 4 days, and examined daily for visible fungal growth. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was applied and macroscopic, microscopic, and physiological characteristics were assessed to identify fungal colonies. 400 pure colonies, belonging to the orders Mucorales and Mortierellales, including the genera Lichtheimia , Rhizopus, Rhizomucor, Mucor, Cunninghamella, and Mortierella, were identified . The genus Rhizopus (35.5%) was the most frequent isolate, followed by Mucor (32.25%) and Rhizomucor (27.5%). The results emphasize the importance of opportunistic fungi in public areas and indicate the risk of exposure for immunocompromised individuals.

  19. Technical note: A method for isolating glycogen granules from ruminal protozoa for further characterization.

    Science.gov (United States)

    Hall, Mary Beth

    2016-03-01

    Evaluation of physical, chemical, and enzymatic hydrolysis characteristics of protozoal glycogen is best performed on a pure substrate to avoid interference from other cell components. A method for isolating protozoal glycogen granules without use of detergents or other potentially contaminating chemicals was developed. Rumen inoculum was incubated anerobically in vitro with glucose. Glycogen-laden protozoa produced in the fermentation, primarily isotrichids, were allowed to sediment in a separatory funnel and were dispensed. The protozoa were processed through repeated centrifugations and sonication to isolate glycogen granules largely free of feed and cellular debris. The final water-insoluble lyophilized product analyzed as 98.3% α-glucan with very rare starch granules and 1.9% protein. Observed losses of glycogen granules during the clean-up process indicate that this procedure should not be used for quantitative assessment of protozoal glycogen from fermentations. Further optimization of this procedure to enhance the amount of glycogen obtained per fermentation may be possible. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  20. Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

    Directory of Open Access Journals (Sweden)

    Haqqani Arsalan S

    2013-01-01

    Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

  1. Spiroketals of Pestalotiopsis fici provide evidence for a biosynthetic hypothesis involving diversified Diels-Alder reaction cascades.

    Science.gov (United States)

    Liu, Ling; Li, Yan; Li, Li; Cao, Ya; Guo, Liangdong; Liu, Gang; Che, Yongsheng

    2013-04-05

    Chloropestolides B-G (1-6), six new metabolites featuring the chlorinated spiro[benzo[d][1,3]dioxine-2,7'-bicyclo[2.2.2]octane]-4,8'-dione (1-3) and spiro[benzo[d][1,3]dioxine-2,1'-naphthalene]-2',4-dione (4-6) skeletons, and their putative biosynthetic precursor dechloromaldoxin (7) were isolated from the scale-up fermentation cultures of the plant endophytic fungus Pestalotiopsis fici . The structures of 1-7 were determined mainly by NMR experiments. The absolute configurations of 1-3 were deduced by analogy to the previously isolated metabolites from the same fungus (9 and 13-18), whereas those of 4, 5, and 7 were assigned by electronic circular dichroism (ECD) calculations. Structurally, the spiroketal skeletons found in 1-3 and 4-6 could be derived from 2,6-dihydroxy-4-methylbenzoic acid with chlorinated bicyclo[2.2.2]oct-2-en-5-one and 4a,5,8,8a-tetrahydronaphthalen-2(1H)-one, respectively. Biogenetically, compounds 1-6 were derived from the same Diels-Alder precursors as the previously isolated 9 and 12-18. In addition, compounds 2 and 3 were proposed as the biosynthetic intermediates of 17 and 16, respectively. Compound 1 was cytotoxic to three human tumor cell lines.

  2. Novel tandem column method for the rapid isolation of radiostrontium from human urine

    International Nuclear Information System (INIS)

    Hawkins, Cory A.; Shkrob, Ilya A.; Mertz, Carol J.; Dietz, Mark L.; Kaminski, Michael D.

    2012-01-01

    Highlights: ► Method for separation and preconcentration of radiostrontium from human urine. ► Recoveries >98%, concentration factor of ca. 50, processing time of nearly 1 h. ► Retention model developed to assist optimization of separations on Diphonix ® column. ► Semi-automated sample preparation device developed. - Abstract: A method has been developed for the isolation of strontium from human urine for subsequent determination in sample volumes as low as 5–20 mL. This method involves the acidification of the sample using methanesulfonic acid and its decolorization using charcoal, treatment of the filtrate with Diphonix ® resin, and subsequent concentration of strontium on Sr resin. Data from retention model simulations provided the initial conditions which were then optimized by actual column separations. Diphonix ® resin was shown to be effective at removing alkali metal ions from the urine matrix under conditions that retain higher valence ions. The suggested processing method provides 99% recovery of Sr 2+ , a concentration factor of 50, and an expected per sample processing time of less than 1 h.

  3. Isolating Trait and Method Variance in the Measurement of Callous and Unemotional Traits.

    Science.gov (United States)

    Paiva-Salisbury, Melissa L; Gill, Andrew D; Stickle, Timothy R

    2017-09-01

    To examine hypothesized influence of method variance from negatively keyed items in measurement of callous-unemotional (CU) traits, nine a priori confirmatory factor analysis model comparisons of the Inventory of Callous-Unemotional Traits were evaluated on multiple fit indices and theoretical coherence. Tested models included a unidimensional model, a three-factor model, a three-bifactor model, an item response theory-shortened model, two item-parceled models, and three correlated trait-correlated method minus one models (unidimensional, correlated three-factor, and bifactor). Data were self-reports of 234 adolescents (191 juvenile offenders, 43 high school students; 63% male; ages 11-17 years). Consistent with hypotheses, models accounting for method variance substantially improved fit to the data. Additionally, bifactor models with a general CU factor better fit the data compared with correlated factor models, suggesting a general CU factor is important to understanding the construct of CU traits. Future Inventory of Callous-Unemotional Traits analyses should account for method variance from item keying and response bias to isolate trait variance.

  4. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  5. Variation in the fumonisin biosynthetic gene cluster in fumonisin-producing and nonproducing black aspergilli.

    Science.gov (United States)

    Susca, Antonia; Proctor, Robert H; Butchko, Robert A E; Haidukowski, Miriam; Stea, Gaetano; Logrieco, Antonio; Moretti, Antonio

    2014-12-01

    The ability to produce fumonisin mycotoxins varies among members of the black aspergilli. Previously, analyses of selected genes in the fumonisin biosynthetic gene (fum) cluster in black aspergilli from California grapes indicated that fumonisin-nonproducing isolates of Aspergillus welwitschiae lack six fum genes, but nonproducing isolates of Aspergillus niger do not. In the current study, analyses of black aspergilli from grapes from the Mediterranean Basin indicate that the genomic context of the fum cluster is the same in isolates of A. niger and A. welwitschiae regardless of fumonisin-production ability and that full-length clusters occur in producing isolates of both species and nonproducing isolates of A. niger. In contrast, the cluster has undergone an eight-gene deletion in fumonisin-nonproducing isolates of A. welwitschiae. Phylogenetic analyses suggest each species consists of a mixed population of fumonisin-producing and nonproducing individuals, and that existence of both production phenotypes may provide a selective advantage to these species. Differences in gene content of fum cluster homologues and phylogenetic relationships of fum genes suggest that the mutation(s) responsible for the nonproduction phenotype differs, and therefore arose independently, in the two species. Partial fum cluster homologues were also identified in genome sequences of four other black Aspergillus species. Gene content of these partial clusters and phylogenetic relationships of fum sequences indicate that non-random partial deletion of the cluster has occurred multiple times among the species. This in turn suggests that an intact cluster and fumonisin production were once more widespread among black aspergilli. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Research of isolated resonances using the average energy shift method for filtered neutron beam

    International Nuclear Information System (INIS)

    Gritzay, O.O.; Grymalo, A.K.; Kolotyi, V.V.; Mityushkin, O.O.; Venediktov, V.M.

    2010-01-01

    This work is devoted to detailed description of one of the research directions in the Neutron Physics Department (NPD), namely, to research of resonance parameters of isolated nuclear level at the filtered neutron beam on the horizontal experimental channel HEC-8 of the WWR-M reactor. Research of resonance parameters is an actual problem nowadays. This is because there are the essential differences between the resonance parameter values in the different evaluated nuclear data library (ENDL) for many nuclei. Research of resonance parameter is possible due to the set of the neutron cross sections received at the same filter, but with the slightly shifted filter average energy. The shift of the filter average energy is possible by several processes. In this work this shift is realized by neutron energy dependence on scattering angle. This method is provided by equipment.

  7. A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes.

    Science.gov (United States)

    Tapia-Tussell, Raul; Lappe, Patricia; Ulloa, Miguel; Quijano-Ramayo, Andrés; Cáceres-Farfán, Mirbella; Larqué-Saavedra, Alfonso; Perez-Brito, Daisy

    2006-05-01

    A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A(260)/A(280)) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.

  8. Methods for the evaluation of antibiotic resistance in Lactobacillus isolated from fermented sausages

    Directory of Open Access Journals (Sweden)

    Hanna Lethycia Wolupeck

    Full Text Available ABSTRACT: The present study aimed to assess the antibiotic resistance in 54 indigenous Lactobacillus plantarum isolated from artisanal fermented sausages. The confirmation of the strain species was performed by multiplex-PCR assay. Antibiotic resistance was assessed by disk diffusion (DD and Minimum Inhibitory Concentration (MIC methods. Of 54 L. plantarum, 44 strains were genotypically confirmed as L. plantarum and 3 as Lactobacillus pentosus. The highest resistance rates were to ampicillin and streptomycin. The highest susceptibility rates were shown to tetracycline, chloramphenicol and penicillin G. None of the strains showed multidrug resistance. Resistance rates by DD and MIC were not different (P>0.05 for ampicillin, chloramphenicol, erythromycin and penicillin G. Future research should assess the genetic mechanisms underlying the phenotypic resistance in Lactobacillus strains to screen the potential probiotic strains for the development of functional meat products.

  9. [Improved methods for researching isolated carotid sinus baroreceptors automatically controlling for sinus pressure].

    Science.gov (United States)

    Wei, Hua; Zhao, Hai-Yan; Liu, Ping; Huang, Hai-Xia; Wang, Wei; Fu, Xiao-Suo; Niu, Wei-Zhen

    2013-01-01

    To develop a system for automatically controlling carotid sinus pressure in the study on baroreceptors. The preparation containing carotid sinus with parts of the connected vessels and carotid sinus nerve (CS-CSN) were isolated and perfused. A critical pressure controlling component (PRE-U, Hoerbiger, Deutschland) dictated by a computer was integrated into the system to clamp the intrasinus pressure. The pressure command and the relevant intrasinus pressure were compared to evaluate the validity of the pressure controlling system. A variety of sinus pressure-controlling patterns, including pulsation, ramp and step pressures, could be achieved accurately by using the system, and the pressure-dependent discharge activities of sinus nerve were confirmed. This system for clamping carotid sinus pressure could realize multiple pressure-controlling patterns and is a useful and flexible pressure controlling method that could applied in the study on mechano-electric transduction of baroreceptors.

  10. Method for the isolation of biologically active monomeric immunoglobulin A from a plasma fraction.

    Science.gov (United States)

    Leibl, H; Tomasits, R; Wolf, H M; Eibl, M M; Mannhalter, J W

    1996-04-12

    A purification method for immunoglobulin A (IgA) yielding monomeric IgA with a purity of over 97% has been developed. This procedure uses ethanol-precipitated plasma (Cohn fraction III precipitate) as the starting material and includes heparin-Sepharose adsorption, dextran sulfate and ammonium sulfate precipitation, hydroxyapatite chromatography, batch adsorption by an anion-exchange matrix and gel permeation. Additional protein G Sepharose treatment leads to an IgA preparation of greater than 99% purity. The isolated IgA presented with an IgA subclass distribution, equivalent to IgA in unfractionated plasma, and was biologically active, as was shown by its ability to down-modulate Haemophilus influenzae-b-induced IL-6 secretion of human monocytes.

  11. Properties of cellulose nanocrystals from oil palm trunk isolated by total chlorine free method.

    Science.gov (United States)

    Lamaming, Junidah; Hashim, Rokiah; Leh, Cheu Peng; Sulaiman, Othman

    2017-01-20

    Cellulose nanocrystals were isolated from oil palm trunk by total chlorine free method. The samples were either water pre-hydrolyzed or non-water pre-hydrolyzed, subjected to soda pulping, acidified and ozone bleached. Cellulose and cellulose nanocrystal (CNC) physical, chemical, thermal properties, and crystallinity index were investigated by composition analysis, scanning electron microscopy, transmission electron microscopy, fourier transform infrared, thermogravimetric analysis and X-ray diffraction. Water pre-hydrolysis reduced lignin (process compared to non-fibrillated of non-water pre-hydrolyzed cellulose. Water pre-hydrolysis improved final CNC crystallinity (up to 75%) compared to CNC without water pre-hydrolysis crystallinity (69%). Cellulose degradation was found to occur during ozone bleaching stage but CNC showed an increase in crystallinity after acid hydrolysis. Thus, oil palm trunk CNC can be potentially applied in pharmaceutical, food, medical and nanocomposites. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Improved method for isolation of lycopsamine from roots of comfrey (Symphytum officinale).

    Science.gov (United States)

    Janes, Damjan; Kalamar, Bostjan; Kreft, Samo

    2012-07-01

    An improved method for the isolation and purification of pyrrolizidine alkaloids from comfrey (Symphytum officinale L.) roots was developed, introducing very fast, selective and ion residue-free reduction of N-oxides followed by ion-exchange chromatography giving a non-aqueous solution of alkaloids, from which solvents can be easily removed. With this procedure the use of large volumes of organic solvents, very slow reduction of N-oxides and input of additional impurities was avoided. Lycopsamine, which proved to be the major alkaloid, was additionally purified by preparative layer chromatography (PLC) and high performance liquid chromatography (HPLC). The identity of the alkaloid was confirmed by (I)H NMR spectroscopy and mass spectrometry.

  13. New method of isolation and perfusion of rat pancreas for phosphorus nucletic resonance study

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, Takehisa; Kanno, Tomio; Murakami, Masataka; Watari, Hiroshi; Seo, Yoshiteru

    1988-02-01

    Phosphorus nuclear magnetic resonance (/sup 31/P-NMR) was applied to measure phosphorus metabolites in the pancreas. Rat pancreatic gland was isolated and perfused with a modified Krebs-Henseleit solution at room temperature (25 deg C). Removal of the duodenum from the pancreas made it possible to record /sup 31/P-NMR spectra only from the pancreatic tissue. /sup 31/P signals of creatine phosphate (PCr), ATP and inorganic phosphate (Pi) were observed in all the glands. In different individual glands, the Signal intensity of PCr varied, but those of ATP were almost the same. The initial levels of all the phosphorus metabolites in individual glands remained almost unchanged during perfusion with the standard solution for 2 hours. When the perfusion was stopped, signal intensity of ATP and PCr decreased while Pi resonance increased and shifted to a higher magnetic field, indicating tissue acidification. On re-perfusion, each resonance and tissue pH were restored to the respective initial resting levels. Continuous stimulation with 0.3 ..mu..M acetylcholine (ACh) or 0.1 nM CCK-8 caused a markedly larger increase in pancreatic exocrine secretion than that observed in the perfused pancreas which was prepared by the previous method. Continuous stimulation with 0.1 ..mu..M ACh caused a marked and sustained increase in pancreatic exocrine secretion. During the stimulation, the resonances of phosphorus energy metabolites and tissue pH remained almost unchanged. These results indicate that the isolated perfused rat pancreas which is prepared by our new method is a useful preparation for analysis of secretion-metabolism coupling in the exocrine pancreas.

  14. Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.

    Science.gov (United States)

    Sun, Wenyi; Yang, Xiaobing; Wang, Xueying; Lin, Xinping; Wang, Yanan; Zhang, Sufang; Luan, Yushi; Zhao, Zongbao K

    2017-07-01

    To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

  15. Antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants.

    Science.gov (United States)

    Gohain, Anwesha; Gogoi, Animesh; Debnath, Rajal; Yadav, Archana; Singh, Bhim P; Gupta, Vijai K; Sharma, Rajeev; Saikia, Ratul

    2015-10-01

    Endophytic actinomycetes are one of the primary groups that share symbiotic relationships with medicinal plants and are key reservoir of biologically active compounds. In this study, six selective medicinal plants were targeted for the first time for endophytic actinomycetes isolation from Gibbon Wild Life Sanctuary, Assam, India, during winter and summer and 76 isolates were obtained. The isolates were found to be prevalent in roots followed by stem and leaves. 16S rRNA gene sequence analysis revealed 16 genera, including rare genera, Verrucosispora, Isoptericola and Kytococcus, which have never been previously reported as endophytic. The genus Streptomyces (66%) was dominant in both seasons. Shannon's diversity index showed that Azadirachta indica (1.49), Rauwolfia serpentina (1.43) and Emblica officinalis (1.24) were relatively good habitat for endophytic actinomycetes. Antimicrobial strains showed prevalence of polyketide synthase (PKS) type-II (85%) followed by PKS type-I (14%) encoded in the genomes. Expression studies showed 12-fold upregulation of PKSII gene in seventh day of incubation for Streptomyces antibioticus (EAAG90). Our results emphasize that the actinomycetes assemblages within plant tissue exhibited biosynthetic systems encoding for important biologically active compounds. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Directory of Open Access Journals (Sweden)

    Yin Zongyi

    Full Text Available Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods. Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets. In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  17. Sea sand disruption method (SSDM) as a valuable tool for isolating essential oil components from conifers.

    Science.gov (United States)

    Dawidowicz, Andrzej L; Czapczyńska, Natalia B

    2011-11-01

    Essential oils are one of nature's most precious gifts with surprisingly potent and outstanding properties. Coniferous oils, for instance, are nowadays being used extensively to treat or prevent many types of infections, modify immune responses, soothe inflammations, stabilize moods, and to help ease all forms of non-acute pain. Given the broad spectrum of usage of coniferous essential oils, a fast, safe, simple, and efficient sample-preparation method is needed in the estimation procedure of essential oil components in fresh plant material. Generally, the time- and energy-consuming steam distillation (SD) is applied for this purpose. This paper will compare SD, pressurized liquid extraction (PLE), matrix solid-phase dispersion (MSPD), and the sea sand disruption method (SSDM) as isolation techniques to obtain aroma components from Scots pine (Pinus sylvestris), spruce (Picea abies), and Douglas fir (Pseudotsuga menziesii). According to the obtained data, SSDM is the most efficient sample preparation method in determining the essential oil composition of conifers. Moreover, SSDM requires small organic solvent amounts and a short extraction time, which makes it an advantageous alternative procedure for the routine analysis of coniferous oils. The superiority of SSDM over MSPD efficiency is ascertained, as there are no chemical interactions between the plant cell components and the sand. This fact confirms the reliability and efficacy of SSDM for the analysis of volatile oil components. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.

  18. Environmental friendly method for the extraction of coir fibre and isolation of nanofibre.

    Science.gov (United States)

    Abraham, Eldho; Deepa, B; Pothen, L A; Cintil, J; Thomas, S; John, M J; Anandjiwala, R; Narine, S S

    2013-02-15

    The objective of this work was to develop an environmental friendly method for the effective utilization of coir fibre by adopting steam pre-treatment. The retting of the coconut bunch makes strong environmental problems which can be avoided by this method. Chemical characterization of the fibre during each processing stages confirmed the increase of cellulose content from raw (40%) to final steam treated fibres (93%). Morphological and dynamic light scattering analyses of the fibres at different processing stages revealed that the isolation of cellulose nano fibres occur in the final step of the process as an aqueous suspension. FT-IR and XRD analysis demonstrated that the treatments lead to the gradual removal of lignin and hemicelluloses from the fibres. The existence of strong lignin-cellulose complex in the raw coir fibre is proved by its enhanced thermal stability. Steam explosion has been proved to be a green method to expand the application areas of coir fibre. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Development of design method of thick rubber bearings for three-dimensional base isolation

    International Nuclear Information System (INIS)

    Yabana, Shuichi; Matuda, Akihiro

    2000-01-01

    Thick rubber bearings as 3-dimensional base isolators have been developed to reduce both horizontal and vertical seismic loads especially for equipment in Fast Breeder Reactors. In this report, a design method of thick rubber bearings is presented. To consider nonlinearity of vertical stiffness affected by vertical stress in the design of thick rubber bearings, Lindley's evaluation method of vertical stiffness is modified as an explicit form of vertical stress. We confirm that the presented method is efficient for design of the thick rubber bearings from comparing between test results and predicted values. Furthermore, rubber bearing tests are conducted with 1/3 scale models to evaluate mechanical properties of thick rubber bearings including ultimate limits. In the tests, horizontal and vertical characteristics of 1/3 scale model are compared with those of 1/6 scale model to discuss scale effect of test specimen. Ultimate limits such as failure shear strain of thick rubber bearings are obtained under various loading conditions. From the test results, we confirm that full scale thick rubber bearing to satisfy requirements is feasible. (author)

  20. Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

    Science.gov (United States)

    Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

    2016-07-01

    Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

  1. Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi.

    Science.gov (United States)

    Serna-Domínguez, María G; Andrade-Michel, Gilda Y; Arredondo-Bernal, Hugo C; Gallou, Adrien

    2018-03-27

    Conventional and commercial methods for isolation of nucleic acids are available for fungal samples including entomopathogenic fungi (EPF). However, there is not a unique optimal method for all organisms. The cell wall structure and the wide range of secondary metabolites of EPF can broadly interfere with the efficiency of the DNA extraction protocol. This study compares three commercial protocols: DNeasy® Plant Mini Kit (Qiagen), Wizard® Genomic DNA Purification Kit (Promega), and Axygen™ Multisource Genomic DNA Miniprep Kit (Axygen) and three conventional methods based on different buffers: SDS, CTAB/PVPP, and CTAB/β-mercaptoethanol versus three cell lysis procedures: liquid nitrogen homogenization and two bead-beating materials (i.e., tungsten-carbide and stainless-steel) for four representative species of EPF (i.e., Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae). Liquid nitrogen homogenization combined with DNeasy® Plant Mini Kit (i.e., QN) or SDS buffer (i.e., SN) significantly improved the yield with a good purity (~1.8) and high integrity (>20,000 bp) of genomic DNA in contrast with other methods, also, these results were better when compared with the two bead-beating materials. The purified DNA was evaluated by PCR-based techniques: amplification of translation elongation factor 1-α (TEF) and two highly sensitive molecular markers (i.e., ISSR and AFLP) with reliable and reproducible results. Despite a variation in yield, purity, and integrity of extracted DNA across the four species of EPF with the different DNA extraction methods, the SN and QN protocols maintained a high-quality of DNA which is required for downstream molecular applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. High Performance Harmonic Isolation By Means of The Single-phase Series Active Filter Employing The Waveform Reconstruction Method

    DEFF Research Database (Denmark)

    Senturk, Osman Selcuk; Hava, Ahmet M.

    2009-01-01

    current sampling delay reduction method (SDRM), a single-phase SAF compensated system provides higher harmonic isolation performance and higher stability margins compared to the system using conventional synchronous reference frame based methods. The analytical, simulation, and experimental studies of a 2...

  3. Expanding the Bioactive Chemical Space of Anthrabenzoxocinones through Engineering the Highly Promiscuous Biosynthetic Modification Steps.

    Science.gov (United States)

    Mei, Xianyi; Yan, Xiaoli; Zhang, Hui; Yu, Mingjia; Shen, Guangqing; Zhou, Linjun; Deng, Zixin; Lei, Chun; Qu, Xudong

    2018-01-19

    Anthrabenzoxocinones (ABXs) including (-)-ABXs and (+)-ABXs are a group of bacterial FabF-specific inhibitors with potent antimicrobial activity of resistant strains. Optimization of their chemical structures is a promising method to develop potent antibiotics. Through biosynthetic investigation, we herein identified and characterized two highly promiscuous enzymes involved in the (-)-ABX structural modification. The promiscuous halogenase and methyltransferase can respectively introduce halogen-modifications into various positions of the ABX scaffolds and methylation to highly diverse substrates. Manipulation of their activity in both of the (-)-ABXs and (+)-ABXs biosyntheses led to the generation of 14 novel ABX analogues of both enantiomers. Bioactivity assessment revealed that a few of the analogues showed significantly improved antimicrobial activity, with the C3-hydroxyl and chlorine substitutions critical for their activity. This study enormously expands the bioactive chemical space of the ABX family and FabF-specific inhibitors. The disclosed broad-selective biosynthetic machineries and structure-activity relationship provide a solid basis for further generation of potent antimicrobial agents.

  4. Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways.

    Science.gov (United States)

    Janevska, Slavica; Tudzynski, Bettina

    2018-01-01

    The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

  5. Expression of carotenoid biosynthetic pathway genes and changes in carotenoids during ripening in tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Namitha, Kanakapura Krishnamurthy; Archana, Surya Narayana; Negi, Pradeep Singh

    2011-04-01

    To study the expression pattern of carotenoid biosynthetic pathway genes, changes in their expression at different stages of maturity in tomato fruit (cv. Arka Ahuti) were investigated. The genes regulating carotenoid production were quantified by a dot blot method using a DIG (dioxigenin) labelling and detection kit. The results revealed that there was an increase in the levels of upstream genes of the carotenoid biosynthetic pathway such as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (Lyt B), phytoene synthase (PSY), phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) by 2-4 fold at the breaker stage as compared to leaf. The lycopene and β-carotene content was analyzed by HPLC at different stages of maturity. The lycopene (15.33 ± 0.24 mg per 100 g) and β-carotene (10.37 ± 0.46 mg per 100 g) content were found to be highest at 5 days post-breaker and 10 days post-breaker stage, respectively. The lycopene accumulation pattern also coincided with the color values at different stages of maturity. These studies may provide insight into devising gene-based strategies for enhancing carotenoid accumulation in tomato fruits.

  6. COMPARATIVE ANALYSIS OF METHODS FOR IDENTIFICATION OF NONTUBERCULOUS MYCOBACTERIA ISOLATED FROM CLINICAL MATERIAL

    Directory of Open Access Journals (Sweden)

    A. V. Lyamin

    2017-01-01

    Full Text Available Recently there has been a significant increase in the incidence of mycobacteriosis, which is due to an increase in the proportion of immunosuppressed patients, the presence of these various comorbid conditions, as well as the improvement of diagnostic methods. Selecting the most accurate method of identification is extremely important in determining treatment strategy of patients. The aim of the study was to conduct a comparative analysis of modern methods of identification NTMB isolated from clinical specimens in 2015 in the Samara region. The work was carried out identification of 78 strains of microorganisms. Laboratory diagnosis was carried out using the DNA hybridization method and MALDI-ToF mass spectrometry. When microbial identification using MALDI-ToF mass spectrometry was isolated 16 strains (20.5% M. kansasii; 11 strains (14.1% M. avium and M. fortuitum; 9 strains (11.5% M. gordonae; strain 3 (3.8% M. peregrinum, M. szulgai, M. chimera intracellulare group, strain 2 (2.6% M. abscessus, M. septicum, M. paragordonae, M. senegalence, 1 strain (1.3% M. chelonae, M. frederiksbergense, M. monacense, M. lentiflavum. By using mass spectrometry, it was identified 15 types NTMB compared with 9 types — by DNA hybridization. Full match identification results was observed only in 45 (57.7% strains of divergent strains were found in 16 (20.5%. Most often when using the DNA hybridization method, discrepancy was detected in slow-growing cultures (9 strains with a predominance of microorganisms identified as M. gordonae. Among the representatives of fast-growing NTMB, seven investigations were identified in the identification, more often among representatives of the M. fortuitum and M. peregrinum groups. Particular attention should be paid to the identification of the M. kansasii strain by a molecular genetic method, which mass spectrometry was defined as M. bovis. Both cultures of M. tuberculosis complex, which were identified by MALDI

  7. Calculating regional tissue volume for hyperthermic isolated limb perfusion: Four methods compared.

    Science.gov (United States)

    Cecchin, D; Negri, A; Frigo, A C; Bui, F; Zucchetta, P; Bodanza, V; Gregianin, M; Campana, L G; Rossi, C R; Rastrelli, M

    2016-12-01

    Hyperthermic isolated limb perfusion (HILP) can be performed as an alternative to amputation for soft tissue sarcomas and melanomas of the extremities. Melphalan and tumor necrosis factor-alpha are used at a dosage that depends on the volume of the limb. Regional tissue volume is traditionally measured for the purposes of HILP using water displacement volumetry (WDV). Although this technique is considered the gold standard, it is time-consuming and complicated to implement, especially in obese and elderly patients. The aim of the present study was to compare the different methods described in the literature for calculating regional tissue volume in the HILP setting, and to validate an open source software. We reviewed the charts of 22 patients (11 males and 11 females) who had non-disseminated melanoma with in-transit metastases or sarcoma of the lower limb. We calculated the volume of the limb using four different methods: WDV, tape measurements and segmentation of computed tomography images using Osirix and Oncentra Masterplan softwares. The overall comparison provided a concordance correlation coefficient (CCC) of 0.92 for the calculations of whole limb volume. In particular, when Osirix was compared with Oncentra (validated for volume measures and used in radiotherapy), the concordance was near-perfect for the calculation of the whole limb volume (CCC = 0.99). With methods based on CT the user can choose a reliable plane for segmentation purposes. CT-based methods also provides the opportunity to separate the whole limb volume into defined tissue volumes (cortical bone, fat and water). Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

    Science.gov (United States)

    McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

    2016-08-06

    FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

  9. Vibration isolation design for periodically stiffened shells by the wave finite element method

    Science.gov (United States)

    Hong, Jie; He, Xueqing; Zhang, Dayi; Zhang, Bing; Ma, Yanhong

    2018-04-01

    Periodically stiffened shell structures are widely used due to their excellent specific strength, in particular for aeronautical and astronautical components. This paper presents an improved Wave Finite Element Method (FEM) that can be employed to predict the band-gap characteristics of stiffened shell structures efficiently. An aero-engine casing, which is a typical periodically stiffened shell structure, was employed to verify the validation and efficiency of the Wave FEM. Good agreement has been found between the Wave FEM and the classical FEM for different boundary conditions. One effective wave selection method based on the Wave FEM has thus been put forward to filter the radial modes of a shell structure. Furthermore, an optimisation strategy by the combination of the Wave FEM and genetic algorithm was presented for periodically stiffened shell structures. The optimal out-of-plane band gap and the mass of the whole structure can be achieved by the optimisation strategy under an aerodynamic load. Results also indicate that geometric parameters of stiffeners can be properly selected that the out-of-plane vibration attenuates significantly in the frequency band of interest. This study can provide valuable references for designing the band gaps of vibration isolation.

  10. Comparison of Composition and Anticaries Effect of Galla Chinensis Extracts with Different Isolation Methods

    Science.gov (United States)

    Huang, Xuelian; Deng, Meng; Liu, Mingdong; Cheng, Lei; Exterkate, R.A.M.; Li, Jiyao; Zhou, Xuedong; Ten Cate, Jacob. M.

    2017-01-01

    Objectives: Galla chinensis water extract (GCE) has been demonstrated to inhibit dental caries by favorably shifting the demineralization/remineralization balance of enamel and inhibiting the biomass and acid formation of dental biofilm. The present study focused on the comparison of composition and anticaries effect of Galla chinensis extracts with different isolation methods, aiming to improve the efficacy of caries prevention. Methods: The composition of water extract (GCE), ethanol extract (eGCE) and commercial tannic acid was compared. High performance liquid chromatography coupled to electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS) analysis was used to analyze the main ingredients. In vitro pH-cycling regime and polymicrobial biofilms model were used to assess the ability of different Galla chinensis extracts to inhibit enamel demineralization, acid formation and biofilm formation. Results: All the GCE, eGCE and tannic acid contained a high level of total phenolics. HPLC-ESI-TOF-MS analysis showed that the main ingredients of GCE were gallic acid (GA), while eGCE mainly contained 4-7 galloylglucopyranoses (GGs) and tannic acid mainly contained 5-10 GGs. Furthermore, eGCE and tannic acid showed a better effect on inhibiting enamel demineralization, acid formation and biofilm formation compared to GCE. Conclusions: Galla chinensis extracts with higher tannin content were suggested to have higher potential to prevent dental caries. PMID:28979574

  11. Improved Simplified Methods for Effective Seismic Analysis and Design of Isolated and Damped Bridges in Western and Eastern North America

    Science.gov (United States)

    Koval, Viacheslav

    The seismic design provisions of the CSA-S6 Canadian Highway Bridge Design Code and the AASHTO LRFD Seismic Bridge Design Specifications have been developed primarily based on historical earthquake events that have occurred along the west coast of North America. For the design of seismic isolation systems, these codes include simplified analysis and design methods. The appropriateness and range of application of these methods are investigated through extensive parametric nonlinear time history analyses in this thesis. It was found that there is a need to adjust existing design guidelines to better capture the expected nonlinear response of isolated bridges. For isolated bridges located in eastern North America, new damping coefficients are proposed. The applicability limits of the code-based simplified methods have been redefined to ensure that the modified method will lead to conservative results and that a wider range of seismically isolated bridges can be covered by this method. The possibility of further improving current simplified code methods was also examined. By transforming the quantity of allocated energy into a displacement contribution, an idealized analytical solution is proposed as a new simplified design method. This method realistically reflects the effects of ground-motion and system design parameters, including the effects of a drifted oscillation center. The proposed method is therefore more appropriate than current existing simplified methods and can be applicable to isolation systems exhibiting a wider range of properties. A multi-level-hazard performance matrix has been adopted by different seismic provisions worldwide and will be incorporated into the new edition of the Canadian CSA-S6-14 Bridge Design code. However, the combined effect and optimal use of isolation and supplemental damping devices in bridges have not been fully exploited yet to achieve enhanced performance under different levels of seismic hazard. A novel Dual-Level Seismic

  12. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik

    1996-01-01

    A kinetic model for the first two steps in the penicillin biosynthetic pathway, i.e. the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS) is proposed. The model is based on Michaelis-Menten type kinetics with non-competitive inhibition of the ACVS by ACV, and competitive inhibition...... of the IPNS by glutathione. The model predicted flux through the pathway corresponds well with the measured rate of penicillin biosynthesis. From the kinetic model the elasticity coefficients and the flux control coefficients are calculated throughout a fed-batch cultivation, and it is found...

  13. Effect of drying methods on the structure, thermo and functional properties of fenugreek (Trigonella foenum graecum) protein isolate.

    Science.gov (United States)

    Feyzi, Samira; Varidi, Mehdi; Zare, Fatemeh; Varidi, Mohammad Javad

    2018-03-01

    Different drying methods due to protein denaturation could alter the functional properties of proteins, as well as their structure. So, this study focused on the effect of different drying methods on amino acid content, thermo and functional properties, and protein structure of fenugreek protein isolate. Freeze and spray drying methods resulted in comparable protein solubility, dynamic surface and interfacial tensions, foaming and emulsifying properties except for emulsion stability. Vacuum oven drying promoted emulsion stability, surface hydrophobicity and viscosity of fenugreek protein isolate at the expanse of its protein solubility. Vacuum oven process caused a higher level of Maillard reaction followed by the spray drying process, which was confirmed by the lower amount of lysine content and less lightness, also more browning intensity. ΔH of fenugreek protein isolates was higher than soy protein isolate, which confirmed the presence of more ordered structures. Also, the bands which are attributed to the α-helix structures in the FTIR spectrum were in the shorter wave number region for freeze and spray dried fenugreek protein isolates that show more possibility of such structures. This research suggests that any drying method must be conducted in its gentle state in order to sustain native structure of proteins and promote their functionalities. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

    Science.gov (United States)

    Davis, Elizabeth; Sloan, Tyler; Aurelius, Krista; Barbour, Angela; Bodey, Elijah; Clark, Brigette; Dennis, Celeste; Drown, Rachel; Fleming, Megan; Humbert, Allison; Glasgo, Elizabeth; Kerns, Trent; Lingro, Kelly; McMillin, MacKenzie; Meyer, Aaron; Pope, Breanna; Stalevicz, April; Steffen, Brittney; Steindl, Austin; Williams, Carolyn; Wimberley, Carmen; Zenas, Robert; Butela, Kristen; Wildschutte, Hans

    2017-06-01

    The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  15. [Comparison of manual and automated (MagNA Pure) nucleic acid isolation methods in molecular diagnosis of HIV infections].

    Science.gov (United States)

    Alp, Alpaslan; Us, Dürdal; Hasçelik, Gülşen

    2004-01-01

    Rapid quantitative molecular methods are very important for the diagnosis of human immunodeficiency virus (HIV) infections, assessment of prognosis and follow up. The purpose of this study was to compare and evaluate the performances of conventional manual extraction method and automated MagNA Pure system, for the nucleic acid isolation step which is the first and most important step in molecular diagnosis of HIV infections. Plasma samples of 35 patients in which anti-HIV antibodies were found as positive by microparticule enzyme immunoassay and confirmed by immunoblotting method, were included in the study. The nucleic acids obtained simultaneously by manual isolation kit (Cobas Amplicor, HIV-1 Monitor Test, version 1.5, Roche Diagnostics) and automated system (MagNA Pure LC Total Nucleic Acid Isolation Kit, Roche Diagnostics), were amplified and detected in Cobas Amplicor (Roche Diagnostics) instrument. Twenty three of 35 samples (65.7%) were found to be positive, and 9 (25.7%) were negative by both of the methods. The agreement between the methods were detected as 91.4%, for qualitative results. Viral RNA copies detected by manual and MagNA Pure isolation methods were found between 76.0-7.590.000 (mean: 487.143) and 113.0-20.300.0000 (mean: 2.174.097) copies/ml, respectively. When both of the overall and individual results were evaluated, the number of RNA copies obtained with automatized system, were found higher than the manual method (p<0.05). Three samples which had low numbers of nucleic acids (113, 773, 857, respectively) with MagNA Pure, yielded negative results with manual method. In conclusion, the automatized MagNA Pure system was found to be a reliable, rapid and practical method for the isolation of HIV-RNA.

  16. Mapping isolated wetlands in a Karst landscape: GIS and remote sensing methods

    Science.gov (United States)

    Isolated wetlands occur in many areas of the United States, and although they are relatively common, they are a resource not yet thoroughly understood by the scientific community. Isolated wetlands have received increased attention recently, due to the 2001 Solid Waste Agency of ...

  17. Scope and status of Russian contribution for analysis methods for seismically isolated nuclear structure

    International Nuclear Information System (INIS)

    Beliayev, V.S.; Vinogradov, V.V.; Guskov, V.D.

    1993-01-01

    In the last few years, we can see in Russia the amplification of interest to problems of seismic isolation for potentially dangerous objects as the most effective way to alleviate the possible damage. This material comprises the data which characterize the level of theoretical design and experimental studying of seismic isolation systems of NPP components and structures. (author)

  18. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

  19. Characterization of ß-Glucans Isolated from Brewer’s Yeast and Dried by Different Methods

    Directory of Open Access Journals (Sweden)

    Vesna Zechner-Krpan

    2010-01-01

    Full Text Available Two different procedures have been used for isolation of water-insoluble ß-glucans from brewer’s yeast: alkaline-acidic isolation (AA and alkaline-acidic isolation with mannoprotein removal (AAM. The obtained ß-glucans were then dried by air-drying, lyophilization and combination of sonication and spray-drying. ß-Glucan preparations obtained by AA and AAM isolations had similar values of dry mass, total polysaccharides, proteins and organic elemental microanalysis. The mass fractions of ß-glucan in total polysaccharides were significantly affected by different isolation procedures. Fourier transform infrared (FTIR spectra of all preparations had the appearance typical for (1→3-ß-D-glucan. Lyophilization and especially air-drying caused a higher degree of agglomeration and changes in ß-glucan microstructure. Sonication followed by spray-drying resulted in minimal structural changes and negligible formation of agglomerates.

  20. Biodiversity of soil-mycoflora of islamabad pakistan using two isolation methods

    International Nuclear Information System (INIS)

    Anjum, F.; Asif, M.; Ayub, N.

    2005-01-01

    The soil microfungal flora of Islamabad were investigated, by using the direct plate and dilution plate methods. A total of 4,225 fungal colonies were isolated from 80 samples collected from Islamabad, Pakistan. The species-composition revealed 66 different species belonging to 20 fungal genera. Among these, Penicillium, being most common and prevalent, was ranked as the dominant genus with respect to the test-sites. Most of the species belong to genera Aspergillus and Penicillium. The maximum number of colonies i.e. 1797 were developed on DRBC, followed by PDA (1320) and the minimum number was recorded on SDA (1108). The results indicate that 4 of these species belong to Mucorales, one to Sphaeriales, one to Coelomycetes and 60 to Hyphomycetes. The most widespread genera were Aspergillus (16 species), Penicillium (15 species) and Acremonium (7 species). The most common species were Aspergillus niger (241 colonies), Cladosporium cladosporioides (219 colonies), Penicillium charlesii (201 colonies), Aspergillus flavus (200 colonies), Penicillium frequantus (198 colonies), Penicillium purpurogenum (178 colonies) and Alternaria alternata (176 colonies). Direct plating and dilution plating with three media, i.e. Potato Dextrose Agar, Dichloran Rose Bengal Agar, and Sabrouad Dextrose Agar, were used in this study. Dilution plating with Dichloran Rose Bengal Chloramphenicol Agar (DRBC) was found to be the most suitable. Other two media i.e. Potato Dextrose Agar (PDA) and Sabrouad Dextrose Agar (SDA) were also tried for cultivation of fungal species with successful results. (author)

  1. Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

    Science.gov (United States)

    Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

    2016-10-20

    In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Various methods for isolation of multipotent human periodontal ligament cells for regenerative medicine.

    Science.gov (United States)

    Tran, Ha Le Bao; Doan, Vu Nguyen; Le, Huong Thi Ngoc; Ngo, Lan Thi Quynh

    2014-08-01

    Periodontal ligament (PDL) is a specialized connective tissue that connects cementum and alveolar bone to maintain and support the teeth in situ and preserve tissue homeostasis. Recent studies have revealed the existence of stem cells in human dental tissues including periodontal ligament that play an important role, not only in the maintenance of the periodontium but also in promoting periodontal regeneration. In this study, human periodontal ligament cells (hPDLCs) were isolated by outgrowth and enzymatic dissociation methods. Expression of surface markers on PDLCs as human mesenchymal stem cells (MSCs) was identified by flow cytometry. In addition, proliferation and differentiation capacity of cultured cells to osteoblasts, adipocytes were evaluated. As a result, we successfully cultured cells from the human periodontal ligament tissues. PDLCs express mesenchymal stem cell (MSC) markers such as CD44, CD73, and CD90 and do not express CD34, CD45, and HLA-DR. PDLCs also possess the multipotential to differentiate into various types of cells, such as osteoblast and adipocytes, in vitro. Therefore, these cells have high potential to serve as materials for tissue engineering, especially dental tissue engineering.

  3. Comparison of Methods for Isolating High Quality DNA and RNA from an Oleaginous Fungus Cunninghamella bainieri Strain 2a1

    Directory of Open Access Journals (Sweden)

    Noor Adila, A. K.

    2007-01-01

    Full Text Available A number of protocols have been reported for efficient fungal DNA and RNA isolation. However, many of these methods are often designed for certain groups or morphological forms of fungi and, in some cases, are species dependent. In this report, we compared four published protocols for DNA isolation from a locally isolated oleaginous fungus, Cunninghamella bainieri strain 2a1. These protocols either involved the use of polyvinyl pyrrolidone (PVP, hexacetyltrimethylammonium bromide (CTAB or without using PVB or CTAB. For RNA isolation, we tested two published protocols, one of which is based on TRI REAGENT (Molecular Research Center, USA and another is simple method employing phenol for RNA extraction and LiCl for precipitation. We found that the protocol involving the use of CTAB produced the highest genomic DNA yield with the best quality compared to other protocols. In the presence of CTAB, unwanted polysaccharides were removed and this method yielded an average amount of 816 ± 12.2 µg DNA/g mycelia with UV absorbance ratios A260/280 and A260/230 of 1.67 ± 0.64 and 1.97 ± 0.23, respectively. The genomic DNA isolated via this protocol is also suitable for PCR amplification and restriction enzyme digestion. As for RNA isolation, the method involving phenol extraction and LiCl precipitation produced the highest yield of RNA with an average amount of 372 ± 6.0 µg RNA/g mycelia. The RNA appears to be relatively pure since it has UV absorbance ratios A260/280 and A260/230 of 1.89 ± 2.00 and 1.99 ± 0.03, respectively. Finally, we have demonstrated that this method could produce RNA of sufficient quality for RT-PCR that amplified a 600 bp fragment of ∆12-fatty acid desaturase gene in C. bainieri.

  4. The Intracellular Localization of the Vanillin Biosynthetic Machinery in Pods of Vanilla planifolia.

    Science.gov (United States)

    Gallage, Nethaji J; Jørgensen, Kirsten; Janfelt, Christian; Nielsen, Agnieszka J Z; Naake, Thomas; Dunski, Eryk; Dalsten, Lene; Grisoni, Michel; Møller, Birger Lindberg

    2018-02-01

    Vanillin is the most important flavor compound in the vanilla pod. Vanilla planifolia vanillin synthase (VpVAN) catalyzes the conversion of ferulic acid and ferulic acid glucoside into vanillin and vanillin glucoside, respectively. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) of vanilla pod sections demonstrates that vanillin glucoside is preferentially localized within the mesocarp and placental laminae whereas vanillin is preferentially localized within the mesocarp. VpVAN is present as the mature form (25 kDa) but, depending on the tissue and isolation procedure, small amounts of the immature unprocessed form (40 kDa) and putative oligomers (50, 75 and 100 kDa) may be observed by immunoblotting using an antibody specific to the C-terminal sequence of VpVAN. The VpVAN protein is localized within chloroplasts and re-differentiated chloroplasts termed phenyloplasts, as monitored during the process of pod development. Isolated chloroplasts were shown to convert [14C]phenylalanine and [14C]cinnamic acid into [14C]vanillin glucoside, indicating that the entire vanillin de novo biosynthetic machinery converting phenylalanine to vanillin glucoside is present in the chloroplast.

  5. A comparison of three methods for the isolation of Arcobacter spp. from retail raw poultry in Northern Ireland

    DEFF Research Database (Denmark)

    Scullion, R.; Harrington, C. S.; Madden, R. H.

    2004-01-01

    Recent evidence suggests that arcobacters, especially Arcobacter butzleri, are potential foodborne pathogens, but standardized detection methods have yet to be established. A study was undertaken to determine which of three isolation methods was the most effective for the isolation of Arcobacter...... spp. from fresh raw poultry. Method I was microaerobic and involved a membrane filtration step followed by plating onto blood agar. Method 2 was also microaerobic and involved enrichment and plating media containing a five-antibiotic cocktail. Method 3 was aerobic and was based on enrichment...... in a charcoal-based broth containing two antibiotics. Retail poultry samples (n = 50) were obtained from supermarkets in Northern Ireland; the European Community license number was recorded to ensure sample diversity. Presumptive arcobacters were identified using genus-specific and species-specific primers...

  6. IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes.

    Science.gov (United States)

    Hadjithomas, Michalis; Chen, I-Min A; Chu, Ken; Huang, Jinghua; Ratner, Anna; Palaniappan, Krishna; Andersen, Evan; Markowitz, Victor; Kyrpides, Nikos C; Ivanova, Natalia N

    2017-01-04

    Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. [Isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris in Yunnan Safari Park].

    Science.gov (United States)

    Cao, Yanru; Jiang, Yi; Li, Youlong; Chen, Xiu; Jin, Rongxian; He, Wenxiang

    2012-07-04

    We studied the isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris by using culture-dependent approaches. Fresh fecal samples of healthy Panthera tigris tigris were collected from Yunnan Safari Park. Pretreatment of the samples, isolation media and inhibitors were tested for actinobacteria isolation. 16S rRNA genes of actinobacteria were sequenced and subjected to phylogenetic analysis. The abundance of culturable actinobacteria was 1.10 x 10(8) cfu/g colony forming units (CFU) per gram of feces (wet weight). We obtained 110 purified cultural actinobacterium strains. The analysis based on 16S rRNA gene sequences showed that these strains were distributed in 10 different families and 12 genera of actinobacteria at least, and most of them were non-filamentous, such as Arthrobacter, Dietzia, Kocuria, Corynebacterium and Microbacterium. Streptomyces was the mainly classical filamentous actinobacteria, and up to 64% of total. Drying and heating up the fecal samples can greatly increase the rate of the actinobacteria. Many kinds of inhibitors and chemical defined media are suitable for isolation of fecal actinobacteria. The culturable actinobacteria are abundant in Panthera tigris tigris feces. Our study found an effective method to isolate animals' fecal actinobacteria and it's useful for studying and exploiting animals' fecal actinobacteria.

  8. Comparison of some storage and isolation methods to recover bluetongue virus from bovine blood.

    OpenAIRE

    Thomas, F C

    1984-01-01

    Bovine blood containing bluetongue virus was stored as whole blood with EDTA (4 degrees C), as washed cellular components (4 degrees C), and as washed cellular components with 10% DMSO (-70 degrees C). Periodic isolation attempts were made over a period of 330 days in four cell lines and embryonating chicken eggs (intravenous inoculation). Bluetongue virus was successfully isolated in all systems from most samples throughout the test period. There appeared to be more variation amongst days of...

  9. New method for the rapid extraction of natural products: efficient isolation of shikimic acid from star anise.

    Science.gov (United States)

    Just, Jeremy; Deans, Bianca J; Olivier, Wesley J; Paull, Brett; Bissember, Alex C; Smith, Jason A

    2015-05-15

    A new, practical, rapid, and high-yielding process for the pressurized hot water extraction (PHWE) of multigram quantities of shikimic acid from star anise (Illicium verum) using an unmodified household espresso machine has been developed. This operationally simple and inexpensive method enables the efficient and straightforward isolation of shikimic acid and the facile preparation of a range of its synthetic derivatives.

  10. A Method for Recruiting Participants from Isolated Islands of Small Island Developing States (SIDS) for Survey Research

    Science.gov (United States)

    Moosa, Sheena; Koopman-Boyden, Peggy

    2016-01-01

    Representing isolated small island communities through social survey research continues to be challenging. We examine a locally developed method to reach and recruit older people (65+ years) for a survey on well-being in the small island developing state of Maldives. The use of messengers to recruit participants is examined in the context of these…

  11. Characterization of single spore isolates of Agaricus bisporus (Lange) Imbach using conventional and molecular methods.

    Science.gov (United States)

    Sharma, Manju; Suman, B C; Gupta, Dharmesh

    2014-10-01

    Strains A-15, S11, S-140, and U3 of Agaricus bisporus (Lange) Imbach, were used as parent strains for raising single spore homokaryotic isolates. Out of total 1,642 single spore isolates, only 36 single spore isolates were homokaryons and exhibited slow mycelial growth rate (≤2.0 mm/day) and appressed colony morphology. All these SSIs failed to produce pinheads in Petri plates even after 65 days of incubation, whereas the strandy slow growing SSIs along with parent strains were able to form the fructification in petriplates after 30 days. Out of 24, six ISSR primers, exhibited scorable bands. In the ISSR fingerprints, single spore isolates, homokaryons, lacked amplification products at multiple loci; they grow slowly and all of them had appressed types of colony morphology. The study revealed losses of ISSR polymorphic patterns in non-fertile homokaryotic single spore isolates compared to the parental control or fertile heterokaryotic single spore isolates.

  12. Comparison of biochemical and molecular methods for the identification of bacterial isolates associated with failed loggerhead sea turtle eggs.

    Science.gov (United States)

    Awong-Taylor, J; Craven, K S; Griffiths, L; Bass, C; Muscarella, M

    2008-05-01

    Comparison of biochemical vs molecular methods for identification of microbial populations associated with failed loggerhead turtle eggs. Two biochemical (API and Microgen) and one molecular methods (16s rRNA analysis) were compared in the areas of cost, identification, corroboration of data with other methods, ease of use, resources and software. The molecular method was costly and identified only 66% of the isolates tested compared with 74% for API. A 74% discrepancy in identifications occurred between API and 16s rRNA analysis. The two biochemical methods were comparable in cost, but Microgen was easier to use and yielded the lowest discrepancy among identifications (29%) when compared with both API 20 enteric (API 20E) and API 20 nonenteric (API 20NE) combined. A comparison of API 20E and API 20NE indicated an 83% discrepancy between the two methods. The Microgen identification system appears to be better suited than API or 16s rRNA analysis for identification of environmental isolates associated with failed loggerhead eggs. Most identification methods are not intended for use with environmental isolates. A comparison of identification systems would provide better options for identifying environmental bacteria for ecological studies.

  13. An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

    Science.gov (United States)

    Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

    2009-11-15

    A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

  14. Intercomparison of analysis methods for seismically isolated nuclear structures. Papers and working materials presented at the 3. research coordination meeting

    International Nuclear Information System (INIS)

    1998-01-01

    The Coordinated research program on Intercomparison of analysis methods for seismically isolated nuclear structures involved participants from Italy, Japan, Republic of Korea, Russia, United Kingdom, USA, EC. The purpose of the meeting was to review the progress on the finite element prediction of the force-deformation behaviour of seismic isolators and to discuss the first set of analytical results for the prediction of the response of base-oscillated structures to earthquake inputs. The intercomparison of predictions of bearing behaviour has identified important unexpected issues requiring deeper investigation

  15. Automated methods for single-stranded DNA isolation and dideoxynucleotide DNA sequencing reactions on a robotic workstation

    International Nuclear Information System (INIS)

    Mardis, E.R.; Roe, B.A.

    1989-01-01

    Automated procedures have been developed for both the simultaneous isolation of 96 single-stranded M13 chimeric template DNAs in less than two hours, and for simultaneously pipetting 24 dideoxynucleotide sequencing reactions on a commercially available laboratory workstation. The DNA sequencing results obtained by either radiolabeled or fluorescent methods are consistent with the premise that automation of these portions of DNA sequencing projects will improve the reproducibility of the DNA isolation and the procedures for these normally labor-intensive steps provides an approach for rapid acquisition of large amounts of high quality, reproducible DNA sequence data

  16. Turnover of radio-iodinated and biosynthetically labelled fibrinogen in rhesus monkeys

    International Nuclear Information System (INIS)

    Moza, A.K.

    1982-01-01

    Successful radio-iodination of monkey fibrinogen using a previously documented method for rabbit fibrinogen is reported. The label was securely bound to fibrinogen without any evidence of polymerisation. Turnover rates and other kinetic parameters of fibrinogen using 125 I-fibrinogen have been compared with those obtained with biosynthetically labelled donor 75 Se-fibrinogen. Both studies yielded identical results. The values for normal monkeys showed a half life of 43.8 +- 1.03 h with 125 I-fibrinogen and 47.15 +- 1.24 with 75 Se-fibrinogen. The turnover rate of endogenous 75 Se-fibrinogen following administration of 75 Se-selenomethionine has also been studied. The half disappearance time value of 100.34 h was much longer than the t1/2 values obtained with either 125 I or 75 Se-fibrinogen. This is believed to be due the staggered input of fibrinogen molecules from the liver. (author)

  17. Evidence for land plant cell wall biosynthetic mechanisms in charophyte green algae

    DEFF Research Database (Denmark)

    Mikkelsen, Maria Dalgaard; Harholt, Jesper; Ulvskov, Peter

    2014-01-01

    in CGA is currently unknown, as no genomes are available, so this study sought to give insight into the evolution of the biosynthetic machinery of CGA through an analysis of available transcriptomes. METHODS: Available CGA transcriptomes were mined for cell wall biosynthesis GTs and compared with GTs...... to colonize land. These cell walls provide support and protection, are a source of signalling molecules, and provide developmental cues for cell differentiation and elongation. The cell wall of land plants is a highly complex fibre composite, characterized by cellulose cross-linked by non......-cellulosic polysaccharides, such as xyloglucan, embedded in a matrix of pectic polysaccharides. How the land plant cell wall evolved is currently unknown: early-divergent chlorophyte and prasinophyte algae genomes contain a low number of glycosyl transferases (GTs), while land plants contain hundreds. The number of GTs...

  18. Characterization of RNA from Exosomes and Other Extracellular Vesicles Isolated by a Novel Spin Column-Based Method

    Science.gov (United States)

    Enderle, Daniel; Spiel, Alexandra; Coticchia, Christine M.; Berghoff, Emily; Mueller, Romy; Schlumpberger, Martin; Sprenger-Haussels, Markus; Shaffer, Jonathan M.; Lader, Eric; Skog, Johan; Noerholm, Mikkel

    2015-01-01

    Exosomes and other extracellular vesicles (commonly referred to as EVs) have generated a lot of attention for their potential applications in both diagnostics and therapeutics. The contents of these vesicles are the subject of intense research, and the relatively recent discovery of RNA inside EVs has raised interest in the biological function of these RNAs as well as their potential as biomarkers for cancer and other diseases. Traditional ultracentrifugation-based protocols to isolate EVs are labor-intensive and subject to significant variability. Various attempts to develop methods with robust, reproducible performance have not yet been completely successful. Here, we report the development and characterization of a spin column-based method for the isolation of total RNA from EVs in serum and plasma. This method isolates highly pure RNA of equal or higher quantity compared to ultracentrifugation, with high specificity for vesicular over non-vesicular RNA. The spin columns have a capacity to handle up to 4 mL sample volume, enabling detection of low-abundance transcripts in serum and plasma. We conclude that the method is an improvement over traditional methods in providing a faster, more standardized way to achieve reliable high quality RNA preparations from EVs in biofluids such as serum and plasma. The first kit utilizing this new method has recently been made available by Qiagen as “exoRNeasy Serum/Plasma Maxi Kit”. PMID:26317354

  19. Comparison of blood RNA isolation methods from samples stabilized in Tempus tubes and stored at a large human biobank.

    Science.gov (United States)

    Aarem, Jeanette; Brunborg, Gunnar; Aas, Kaja K; Harbak, Kari; Taipale, Miia M; Magnus, Per; Knudsen, Gun Peggy; Duale, Nur

    2016-09-01

    More than 50,000 adult and cord blood samples were collected in Tempus tubes and stored at the Norwegian Institute of Public Health Biobank for future use. In this study, we systematically evaluated and compared five blood-RNA isolation protocols: three blood-RNA isolation protocols optimized for simultaneous isolation of all blood-RNA species (MagMAX RNA Isolation Kit, both manual and semi-automated protocols; and Norgen Preserved Blood RNA kit I); and two protocols optimized for large RNAs only (Tempus Spin RNA, and Tempus 6-port isolation kit). We estimated the following parameters: RNA quality, RNA yield, processing time, cost per sample, and RNA transcript stability of six selected mRNAs and 13 miRNAs using real-time qPCR. Whole blood samples from adults (n = 59 tubes) and umbilical cord blood (n = 18 tubes) samples collected in Tempus tubes were analyzed. High-quality blood-RNAs with average RIN-values above seven were extracted using all five RNA isolation protocols. The transcript levels of the six selected genes showed minimal variation between the five protocols. Unexplained differences within the transcript levels of the 13 miRNA were observed; however, the 13 miRNAs had similar expression direction and they were within the same order of magnitude. Some differences in the RNA processing time and cost were noted. Sufficient amounts of high-quality RNA were obtained using all five protocols, and the Tempus blood RNA system therefore seems not to be dependent on one specific RNA isolation method.

  20. Production, Isolation and Radiolabeling Methods for 211AT- Labeling of Biomolecules

    International Nuclear Information System (INIS)

    Wilbur, D.S.; Hamlin, D.K.; Chyan, M.

    2009-01-01

    Targeted alpha therapy with 211 At-labeled compounds holds great promise for treatment of cancer, particularly compartmentalized cancer (e.g. ovarian cancer), minimal residual cancer after surgery and metastatic disease. Unfortunately, 211 At has limited availability and, due to its unique nature, has the potential to be readily dissociated from the cancer-targeting agents used in vivo. Finding methods to circumvent these two problems has occupied a large amount of our efforts over the past few years. 211 At is produced at the University of Washington on a Scanditronix MC-50 using a 28 MeV alpha beam. Our initial preclinical studies were conducted using a small target assembly with irradiations of a 10 □ A alpha beam, but our desire to ultimately conduct clinical studies led to the design and installation of a new target assembly that had much larger irradiation surface and would withstand beam energies of 50 □A or more. Prior to this upgrade, 211 At was efficiently isolated (60-80%) from the irradiated aluminum-backed bismuth targets by dry distillation at 650 o C. However, the dry distillation method gave low recovery yields (e.g. 10-40%) when the much larger new targets were used. After some attempts to improve the distillation yields, we have more recently conducted a wet chemistry approach to the 211 At isolation. While this method still needs to be optimized, it has provided good recovery (60-90%) of the 211 At. Our radiolabeling methods have undergone a similar transition in the past few years. Until recently our 211 At studies were limited to the use of intact monoclonal antibodies (MAb) labeled using conjugates containing aryl-astatine derivatives due to the deastatination of more rapidly metabolized targeting biomolecules. This limitation made it all but impossible to label important biomolecules such as MAb fragments, engineered proteins, peptides and small molecules. This critical shortcoming of labeling methods for 211 At led to our investigating

  1. Detection of Chloramphenicol Resistance Genes (cat in Clinical Isolates of Pseudomonas aeruginosa with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-12-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic Gram negative bacteria, which may cause infection in eyes, ears, skin, bones, central nervous system, gastrointestinal tract, circulatory system, heart, respiratory system, and urinary tract. Recently, chloramphenicol is no longer used as the main option of the therapy due of its resistance case. The aim of this research was to detect the presence of gene which is responsible to chloramphenicol resistance in clinical isolates of P.aeruginosa. These bacteria isolated from pus of external otitis patients in Hasan Sadikin Hospital in Bandung City. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were performed to detect this resistance gene. Electropherogram from PCR products showed that the chloramphenicol resistance in clinical isolates of P. aeruginosa was caused by cat gene (317 bp. Based on this research, cat gene may be used to detect the chloramphenicol resistance in patients with external ostitis.

  2. A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP.

    Science.gov (United States)

    Kim, C S; Lee, C H; Shin, J S; Chung, Y S; Hyung, N I

    1997-03-01

    Because DNA degradation is mediated by secondary plant products such as phenolic terpenoids, the isolation of high quality DNA from plants containing a high content of polyphenolics has been a difficult problem. We demonstrate an easy extraction process by modifying several existing ones. Using this process we have found it possible to isolate DNAs from four fruit trees, grape (Vitis spp.), apple (Malus spp.), pear (Pyrus spp.) and persimmon (Diospyros spp.) and four species of conifer, Pinus densiflora, Pinus koraiensis,Taxus cuspidata and Juniperus chinensis within a few hours. Compared with the existing method, we have isolated high quality intact DNAs (260/280 = 1.8-2.0) routinely yielding 250-500 ng/microl (total 7.5-15 microg DNA from four to five tissue discs).

  3. Molecular profiles of Venezuelan isolates of Trypanosoma sp. by random amplified polymorphic DNA method.

    Science.gov (United States)

    Perrone, T M; Gonzatti, M I; Villamizar, G; Escalante, A; Aso, P M

    2009-05-12

    Nine Trypanosoma sp. Venezuelan isolates, initially presumed to be T. evansi, were collected from three different hosts, capybara (Apure state), horse (Apure state) and donkey (Guarico state) and compared by the random amplification polymorphic DNA technique (RAPD). Thirty-one to 46 reproducible fragments were obtained with 12 of the 40 primers that were used. Most of the primers detected molecular profiles with few polymorphisms between the seven horse, capybara and donkey isolates. Quantitative analyses of the RAPD profiles of these isolates revealed a high degree of genetic conservation with similarity coefficients between 85.7% and 98.5%. Ten of the primers generated polymorphic RAPD profiles with two of the three Trypanosoma sp. horse isolates, namely TeAp-N/D1 and TeGu-N/D1. The similarity coefficient between these two isolates and the rest, ranged from 57.9% to 68.4% and the corresponding dendrogram clustered TeAp-N/D1 and Te Gu-N/D1 in a genetically distinct group.

  4. The flavonoid biosynthetic pathway in plants: function and evolution

    International Nuclear Information System (INIS)

    Koes, R.E.; Quattrocchio, F.; Mol, J.N.M.

    1994-01-01

    Flavonoids are a class of low molecular weight phenolic compounds that is widely distributed in the plant kingdom. They exhibit a diverse spectrum of biological functions and play an important role in the interaction between plants and their environment. Flavonoids not only protect the plant from the harmful effects of UV irradiation but also play a crucial role in the sexual reproduction process. A special class of flavonoid polymers, the tannins, plays a structural role in the plant. Yet other classes of flavonoids, flavonols and anthocyanins, have been implicated in the attraction of pollinators. Certain flavonoids participate in the interaction between plants and other organisms such as symbiotic bacteria and parasites. This raises the intriguing question as to how these different compounds arose and evolved. Based on taxonomy and molecular analysis of gene expression patterns it is possible to deduce a putative sequence of acquisition of the different branches of the biosynthetic pathway and their regulators. (author)

  5. The flavonoid biosynthetic pathway in plants: function and evolution

    Energy Technology Data Exchange (ETDEWEB)

    Koes, R. E.; Quattrocchio, F.; Mol, J. N.M. [Department of Genetics, Institute for Molecular Biological Sciences, Vrije Universiteit, BioCentrum Amsterdam, De Boelelaan 1087, 1081HV, Amsterdam (Netherlands)

    1994-07-01

    Flavonoids are a class of low molecular weight phenolic compounds that is widely distributed in the plant kingdom. They exhibit a diverse spectrum of biological functions and play an important role in the interaction between plants and their environment. Flavonoids not only protect the plant from the harmful effects of UV irradiation but also play a crucial role in the sexual reproduction process. A special class of flavonoid polymers, the tannins, plays a structural role in the plant. Yet other classes of flavonoids, flavonols and anthocyanins, have been implicated in the attraction of pollinators. Certain flavonoids participate in the interaction between plants and other organisms such as symbiotic bacteria and parasites. This raises the intriguing question as to how these different compounds arose and evolved. Based on taxonomy and molecular analysis of gene expression patterns it is possible to deduce a putative sequence of acquisition of the different branches of the biosynthetic pathway and their regulators. (author)

  6. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts

    DEFF Research Database (Denmark)

    Nielsen, Agnieszka Janina Zygadlo; Mellor, Silas Busck; Vavitsas, Konstantinos

    2016-01-01

    The chloroplasts found in plants and algae, and photosynthetic microorganisms such as cyanobacteria, are emerging hosts for sustainable production of valuable biochemicals, using only inorganic nutrients, water, CO2 and light as inputs. In the past decade, many bioengineering efforts have focused...... on metabolic engineering and synthetic biology in the chloroplast or in cyanobacteria for the production of fuels, chemicals, as well as complex, high-value bioactive molecules. Biosynthesis of all these compounds can be performed in photosynthetic organelles/organisms by heterologous expression...... of chloroplasts and cyanobacteria as biosynthetic compartments and hosts, and we estimate the production levels to be expected from photosynthetic hosts in light of the fraction of electrons and carbon that can potentially be diverted from photosynthesis. The supply of reducing power, in the form of electrons...

  7. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Haselkorn, Tamara; Bunce, Michael

    2007-01-01

    . Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase "blocks" during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits....... These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids...

  8. In silico exploration of Red Sea Bacillus genomes for natural product biosynthetic gene clusters

    KAUST Repository

    Othoum, Ghofran K

    2018-05-22

    BackgroundThe increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions.ResultsWe report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species.ConclusionsB. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems.

  9. Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

    Directory of Open Access Journals (Sweden)

    Valerio Mori

    Full Text Available NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT, which in mammals comprises three distinct isozymes, and NAD synthetase (NADS. First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide. In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

  10. Evolution and Diversity of Biosynthetic Gene Clusters in Fusarium

    Directory of Open Access Journals (Sweden)

    Koen Hoogendoorn

    2018-06-01

    Full Text Available Plant pathogenic fungi in the Fusarium genus cause severe damage to crops, resulting in great financial losses and health hazards. Specialized metabolites synthesized by these fungi are known to play key roles in the infection process, and to provide survival advantages inside and outside the host. However, systematic studies of the evolution of specialized metabolite-coding potential across Fusarium have been scarce. Here, we apply a combination of bioinformatic approaches to identify biosynthetic gene clusters (BGCs across publicly available genomes from Fusarium, to group them into annotated families and to study gain/loss events of BGC families throughout the history of the genus. Comparison with MIBiG reference BGCs allowed assignment of 29 gene cluster families (GCFs to pathways responsible for the production of known compounds, while for 57 GCFs, the molecular products remain unknown. Comparative analysis of BGC repertoires using ancestral state reconstruction raised several new hypotheses on how BGCs contribute to Fusarium pathogenicity or host specificity, sometimes surprisingly so: for example, a gene cluster for the biosynthesis of hexadehydro-astechrome was identified in the genome of the biocontrol strain Fusarium oxysporum Fo47, while being absent in that of the tomato pathogen F. oxysporum f.sp. lycopersici. Several BGCs were also identified on supernumerary chromosomes; heterologous expression of genes for three terpene synthases encoded on the Fusarium poae supernumerary chromosome and subsequent GC/MS analysis showed that these genes are functional and encode enzymes that each are able to synthesize koraiol; this observed functional redundancy supports the hypothesis that localization of copies of BGCs on supernumerary chromosomes provides freedom for evolutionary innovations to occur, while the original function remains conserved. Altogether, this systematic overview of biosynthetic diversity in Fusarium paves the way for

  11. Expression of eicosanoid biosynthetic and catabolic enzymes in peritoneal endometriosis.

    Science.gov (United States)

    Lousse, J-C; Defrère, S; Colette, S; Van Langendonckt, A; Donnez, J

    2010-03-01

    Increased peritoneal eicosanoid concentrations have been reported in endometriosis patients and might be important in disease-associated pain and inflammation. Here, we evaluated the expression of key biosynthetic and catabolic enzymes involved in this abnormal eicosanoid production in peritoneal macrophages and endometriotic lesions. Peritoneal macrophages, endometriotic lesions and matched eutopic endometrium were collected from endometriosis patients (n = 40). Peritoneal macrophages and eutopic endometrium samples were also collected from disease-free women (n = 25). Expression of type IIA secretory phospholipase A(2) (sPLA(2)-IIA), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 5-lipoxygenase (5-LO) was quantified by real-time PCR, and these five key enzymes were localized by immunohistochemistry. sPLA(2)-IIA, COX-2 and mPGES-1 mRNA was significantly increased in peritoneal macrophages of endometriosis patients compared with controls (P = 0.006, P = 0.016 and P = 0.025, respectively). In endometriosis patients, sPLA(2)-IIA, mPGES-1 and 15-PGDH mRNA was significantly enhanced in peritoneal lesions compared with matched eutopic endometrium (P endometriosis group compared with controls (P = 0.023). Finally, sPLA(2)-IIA, COX-2, mPGES-1 and 15-PGDH immunostaining was found mainly in endometrial glands, whereas 5-LO was distributed throughout the glands and stroma. Our study highlights an imbalance between eicosanoid biosynthesis and degradation in endometriosis patients. Both peritoneal macrophages and endometriotic lesions may be involved. Research into new molecules inhibiting biosynthetic enzymes (such as sPLA(2)-IIA and mPGES-1) and/or activating catabolic enzymes (such as 15-PGDH) may prove to be a major field of investigation in the development of targeted medical therapies.

  12. The Isolation of Pure Populations of Neurons by Laser Capture Microdissection: Methods and Application in Neuroscience.

    Science.gov (United States)

    Morris, Renée; Mehta, Prachi

    2018-01-01

    In mammals, the central nervous system (CNS) is constituted of various cellular elements, posing a challenge to isolating specific cell types to investigate their expression profile. As a result, tissue homogenization is not amenable to analyses of motor neurons profiling as these represent less than 10% of the total spinal cord cell population. One way to tackle the problem of tissue heterogeneity and obtain meaningful genomic, proteomic, and transcriptomic profiling is to use laser capture microdissection technology (LCM). In this chapter, we describe protocols for the capture of isolated populations of motor neurons from spinal cord tissue sections and for downstream transcriptomic analysis of motor neurons with RT-PCR. We have also included a protocol for the immunological confirmation that the captured neurons are indeed motor neurons. Although focused on spinal cord motor neurons, these protocols can be easily optimized for the isolation of any CNS neurons.

  13. Phenotypic methods for detection of various β-lactamases in Gram-negative clinical isolates: Need of the hour

    Directory of Open Access Journals (Sweden)

    Neena V Nagdeo

    2012-01-01

    Full Text Available Background: Many clinical laboratories have problems detecting various β-lactamases. Confusion exists about the importance of these resistance mechanisms, optimal test methods, and appropriate reporting conventions. It is more imperative to use various phenotypic methods for detection of various β-lactamases in routine microbiology laboratory on day-to-day basis to prevent antimicrobial resistance by evidence-based judicious use of antimicrobials. Aims: In view of the multidrug-resistant organisms being reported world over, we planned a cross-sectional prospective analytical study to determine resistance mechanism by various β-lactamases in Gram-negative clinical isolates using various phenotypic methods. Materials and Methods: All nonrepeat, nonenteric clinical isolates of Gram-negative bacilli, resistant to at least two third-generation cephalosporins, were first screened by Novel disc placement method, and isolates showing multiple mechanisms of resistance and reduced zone of inhibition for imipenem were further confirmed for AmpC and metallo β-lactamases. Statistical Analysis: All the data was managed and analyzed in Microsoft Excel. Results: Out of 807 isolates tested, as many as 795 (98.51% revealed the presence of extended-spectrum β-lactamases (ESBLs. Only 10 isolates of Escherichia coli and 2 of Klebsiella pneumoniae did not show production of ESBL. A total of 450 (55.76% isolates produced single enzyme,while 345 (42.75% strains revealed multiple enzyme production simultaneously. Only ESBL production was seen in 315 (39.03% strains, only AmpC in 75 (9.29% and only MBL in 60 (7.44% strains, while ESBL and AmpC together were seen in 219 (27.14% and AmpC plus MBL in 92 (11.40% strains. However, ESBL plus MBL were never observed together. All three enzymes were simultaneously detected in 34 (4.21% strains. Conclusion: This innovative method of disc placement makes it easy, affordable, and reliable method for routine use by basic

  14. Comparative study of different methods used to isolate Trypanosoma cruz i for defibrinated blood of irradiated mice

    International Nuclear Information System (INIS)

    Fontes, G.; Romanha, A.J.; Brener, Z.

    1991-01-01

    Different methods are being used for the isolation and purification of Trypanosoma cruzi blood forms from infected vertebrate hosts. In this study, we compare four of these methods (differential centrifugation, Ficoll-Hypaque, Histopaque 1077 and metrizamide) in terms of parasite recovery rates, contamination with cells, duration of the process and role of host irradiation. Male albino Swiss mice irradiated in a Gamma Cell 220(500 rads) were inoculated with C L and V-10 T.cruzi strains and bled at the peak of parasitemia. Infected defibrinated blood was then used for the isolation. Although all methods permitted the recovery of viable trypomastigotes, the best results were obtained with Ficoll-Hypaque and Histopaque 1077. Recovery rates ranged between 71% to 88% and parasite-enriched preparations were obtained in approximately 75 min. Irradiation and blood defibrination drastically reduced platelet and leukocyte contamination of the preparations. (author)

  15. TX-RX isolation method based on polarization diversity, spatial diversity and TX beamforming

    DEFF Research Database (Denmark)

    Foroozanfard, Ehsan; Carvalho, Elisabeth De; Pedersen, Gert F.

    2016-01-01

    In this paper, the feasibility of an antenna isolation technique based on null-steer beamforming, polarization diversity and spatial diversity is investigated. The proposed system consists of six patch antennas which are fed by a feeding network to obtain a null-steer beamformer. To achieve spatial...... diversity, antenna elements are located on two layers, facing in a different direction. Moreover, the antenna elements in two layers use different polarization. The measured results of the antenna system present a high TX-RX isolation in the order of 70 dB which shows the feasibility of such a system...

  16. Thermally-isolated silicon-based integrated circuits and related methods

    Science.gov (United States)

    Wojciechowski, Kenneth; Olsson, Roy H.; Clews, Peggy J.; Bauer, Todd

    2017-05-09

    Thermally isolated devices may be formed by performing a series of etches on a silicon-based substrate. As a result of the series of etches, silicon material may be removed from underneath a region of an integrated circuit (IC). The removal of the silicon material from underneath the IC forms a gap between remaining substrate and the integrated circuit, though the integrated circuit remains connected to the substrate via a support bar arrangement that suspends the integrated circuit over the substrate. The creation of this gap functions to release the device from the substrate and create a thermally-isolated integrated circuit.

  17. Method of making thermally-isolated silicon-based integrated circuits

    Science.gov (United States)

    Wojciechowski, Kenneth; Olsson, Roy; Clews, Peggy J.; Bauer, Todd

    2017-11-21

    Thermally isolated devices may be formed by performing a series of etches on a silicon-based substrate. As a result of the series of etches, silicon material may be removed from underneath a region of an integrated circuit (IC). The removal of the silicon material from underneath the IC forms a gap between remaining substrate and the integrated circuit, though the integrated circuit remains connected to the substrate via a support bar arrangement that suspends the integrated circuit over the substrate. The creation of this gap functions to release the device from the substrate and create a thermally-isolated integrated circuit.

  18. Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods

    Directory of Open Access Journals (Sweden)

    Sahar Kianipour

    2018-01-01

    Full Text Available Background: Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species. Materials and Methods: Polymerase chain reaction (PCR-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media. Results: Fifty-nine (49.2% yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6% of C. albicans/dubliniensis complex and 12 (41.4% of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9% were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients. Conclusion: Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.

  19. The Role of Isolation Methods on a Nanoscale Surface Structure and Its Effect on the Size of Exosomes

    Directory of Open Access Journals (Sweden)

    JungReem Woo

    2016-06-01

    Full Text Available Exosomes are ~100 nanometre diameter vesicles secreted by mammalian cells. These emerging disease biomarkers carry nucleic acids, proteins and lipids specific to the parental cells that secrete them. Exosomes are typically isolated in bulk by ultracentrifugation, filtration or immu‐ noaffinity precipitation for downstream proteomic, genomic, or lipidomic analysis. However, the structural properties and heterogeneity of isolated exosomes at the single vesicle level are not well characterized due to their small size. In this paper, by using high-resolution atomic force microscope imaging, we show the nanoscale mor‐ phology and structural heterogeneity in exosomes derived from U87 cells. Quantitative assessment of single exosomes reveals nanoscale variations in morphology, surface roughness and counts isolated by ultracentrifugation (UC and immunoaffinity (IA purification. Both methods produce intact globular, 30-120 nm sized vesicles when imaged under fluid and in air. However, IA exosomes had higher surface roughness and bimodal size population compared to UC exosomes. The study highlights the differences in size and surface topography of exosomes purified from a single cell type using different isolation methods.

  20. A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

    Science.gov (United States)

    Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

    2017-09-01

    Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli

    DEFF Research Database (Denmark)

    Brown, S; Brickman, E R; Beckwith, J

    1981-01-01

    We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon inser...

  2. Hydrologic connectivity between geographically isolated wetlands and surface water systems: A review of select modeling methods

    Science.gov (United States)

    Heather E. Golden; Charles R. Lane; Devendra M. Amatya; Karl W. Bandilla; Hadas Raanan Kiperwas Kiperwas; Christopher D. Knightes; Herbert. Ssegane

    2014-01-01

    Geographically isolated wetlands (GIW), depressional landscape features entirely surrounded by upland areas, provide a wide range of ecological functions and ecosystem services for human well-being. Current and future ecosystem management and decision-making rely on a solid scientific understanding of how hydrologic processes affect these important GIW services and...

  3. A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood.

    Science.gov (United States)

    Monti, Manuela; Imberti, Barbara; Bianchi, Niccolò; Pezzotta, Anna; Morigi, Marina; Del Fante, Claudia; Redi, Carlo Alberto; Perotti, Cesare

    2017-09-01

    Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

  4. New method of plant mitochondria isolation and sub-fractionation for proteomic analyses

    Czech Academy of Sciences Publication Activity Database

    Hájek, Tomáš; Honys, David; Čapková, Věra

    2004-01-01

    Roč. 167, č. 3 (2004), s. 389-395 ISSN 0168-9452 R&D Projects: GA MŠk LZ1K03018 Institutional research plan: CEZ:AV0Z5038910 Keywords : plant mitochondria isolation * sub-fractionation * protein analysis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.389, year: 2004

  5. Optimization of HPLC method for the isolation of Hypericum perforatum L. methanol extract

    Directory of Open Access Journals (Sweden)

    Stamenković, J.

    2013-12-01

    Full Text Available St. John's Wort (Hypericum perforatum L. is one of the most studied plant species in the family Hypericaceae. The aim of this study was the identification of the constituents of methanol extract of H. perforatum and optimization of conditions for their isolation. The main components of the methanol extract were isolated on preparative ZORBAX Eclipse XDB C18 column with solvent system consisting of methanol and 1x10-2 M ammonium acetate in water. Constituents of the extract were identified by comparing their retention times with the retention times of the standards, with the literature data and the UV spectra. By varying the conditions of chromatography, the optimal conditions for isolation of the methanol extract constituents were determined: mobile phase consisting of methanol and 1x10-2 M ammonium acetate in water in ratio 1 : 1, sample concentration 100 mg/mL, sample volume 30 µL, flow 2 mL/min. Under these conditions 7 components of the methanol extract were isolated.

  6. Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp.

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    Sandra Fresquet-Corrales

    Full Text Available Proanthocyanidins (PAs, or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (- catechin/g FW and 228.5 nmol (- epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloat-safe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass are discussed.

  7. Analysis of occludin trafficking, demonstrating continuous endocytosis, degradation, recycling and biosynthetic secretory trafficking.

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    Sarah J Fletcher

    Full Text Available Tight junctions (TJs link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes. By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.

  8. Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods.

    Science.gov (United States)

    Kianipour, Sahar; Ardestani, Mohammad Emami; Dehghan, Parvin

    2018-01-01

    Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species. Polymerase chain reaction (PCR)-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL) samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media. Fifty-nine (49.2%) yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6%) of C. albicans / dubliniensis complex and 12 (41.4%) of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9%) were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients. Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.

  9. COMPARATIVE EVALUATION OF CONVENTIONAL VERSUS RAPID METHODS FOR AMPLIFIABLE GENOMIC DNA ISOLATION OF CULTURED Azospirillum sp. JG3

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    Stalis Norma Ethica

    2013-12-01

    Full Text Available As an initial attempt to reveal genetic information of Azospirillum sp. JG3 strain, which is still absence despite of the strains' ability in producing valued enzymes, two groups of conventional methods: lysis-enzyme and column-kit; and two rapid methods: thermal disruption and intact colony were evaluated. The aim is to determine the most practical method for obtaining high-grade PCR product using degenerate primers as part of routine-basis protocols for studying the molecular genetics of the Azospirillal bacteria. The evaluation includes the assessment of electrophoresis gel visualization, pellet appearance, preparation time, and PCR result of extracted genomic DNA from each method. Our results confirmed that the conventional methods were more superior to the rapid methods in generating genomic DNA isolates visible on electrophoresis gel. However, modification made in the previously developed DNA isolation protocol giving the simplest and most rapid method of all methods used in this study for extracting PCR-amplifiable DNA of Azospirillum sp. JG3. Intact bacterial cells (intact colony loaded on electrophoresis gel could present genomic DNA band, but could not be completely amplified by PCR without thermal treatment. It can also be inferred from our result that the 3 to 5-min heating in dH2O step is critical for the pre-treatment of colony PCR of Azospirillal cells.

  10. Molecular and Biochemical Analysis of Chalcone Synthase from Freesia hybrid in flavonoid biosynthetic pathway.

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    Wei Sun

    Full Text Available Chalcone synthase (CHS catalyzes the first committed step in the flavonoid biosynthetic pathway. In this study, the cDNA (FhCHS1 encoding CHS from Freesia hybrida was successfully isolated and analyzed. Multiple sequence alignments showed that both the conserved CHS active site residues and CHS signature sequence were found in the deduced amino acid sequence of FhCHS1. Meanwhile, crystallographic analysis revealed that protein structure of FhCHS1 is highly similar to that of alfalfa CHS2, and the biochemical analysis results indicated that it has an enzymatic role in naringenin biosynthesis. Moreover, quantitative real-time PCR was performed to detect the transcript levels of FhCHS1 in flowers and different tissues, and patterns of FhCHS1 expression in flowers showed significant correlation to the accumulation patterns of anthocyanin during flower development. To further characterize the functionality of FhCHS1, its ectopic expression in Arabidopsis thaliana tt4 mutants and Petunia hybrida was performed. The results showed that overexpression of FhCHS1 in tt4 mutants fully restored the pigmentation phenotype of the seed coats, cotyledons and hypocotyls, while transgenic petunia expressing FhCHS1 showed flower color alteration from white to pink. In summary, these results suggest that FhCHS1 plays an essential role in the biosynthesis of flavonoid in Freesia hybrida and may be used to modify the components of flavonoids in other plants.

  11. Location, formation and biosynthetic regulation of cellulases in the gliding bacteria Cytophaga hutchinsonii

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    Elijah Johnson

    2006-01-01

    Full Text Available An analysis of the recently published genome sequence of Cytophagahutchinsonii revealed an unusual collection of genes for an organism that can attackcrystalline cellulose. Consequently, questions were being raised by cellulase scientists, as towhat mechanism this organism uses to degrade its insoluble substrates. Cellulose, being ahighly polymeric compound and insoluble in water, cannot enter the cell walls ofmicroorganisms. Cellulose-degrading enzymes have therefore to be located on the surface ofthe cell wall or released extracellularly. The location of most cellulase enzymes has beenstudied. However, basic information on C. hutchinsonii cellulases is almost non-existent. Inthe present study, the location, formation and biosynthetic regulation of cellulases in C.hutchinsonii were demonstrated on different substrates. Various fractions isolated from C.hutchinsonii after cell rupture were assayed for carboxymethyl-cellulase activity (CMC.The cellulases were found to be predominantly cell-free during active growth on solka-flok,although 30% of activity was recorded on cell-bound enzymes. Relatively little CM-cellulase was formed when cells were grown on glucose and cellobiose. Apparently glucoseor labile substrates such as cellobiose seem to repress the formation of CM-cellulase. Thesefindings should provide some insight into possible hydrolysis mechanisms by C.hutchinsonii.

  12. A simple cleanup method for the isolation of nitrate from natural water samples for O isotopes analysis

    International Nuclear Information System (INIS)

    Haberhauer, G.; Blochberger, K.

    1999-09-01

    The analysis of O-isotopic composition of nitrate has many potential applications in studies of environmental processes. O-isotope nitrate analysis require sample free of other oxygen-containing compounds. More than 100 % of non-NO 3 - oxygen relative to NO 3 - oxygen can still be found in forest soil water samples after cleanup if improper cleanup strategies, e.g., adsorption onto activated carbon, are used. Such non-NO 3 - oxygen compounds will bias O-isotropic data. Therefore, an efficient cleanup method was developed to isolate nitrate from natural water samples. In a multistep cleanup procedure using adsorption onto water-insoluble poly(vinylpyrrolidone), removal of almost all other oxygen-containing compounds, such as fulvic acids, and isolation of nitrate was achieved. The method supplied samples free of non-NO 3 - oxygen which can be directly combusted to CO 2 for subsequent O-isotope analysis. (author)

  13. Evaluation of different continuous cell lines in the isolation of mumps virus by the shell vial method from clinical samples

    Science.gov (United States)

    Reina, J; Ballesteros, F; Mari, M; Munar, M

    2001-01-01

    Aims—To compare prospectively the efficacy of the Vero, LLC-MK2, MDCK, Hep-2, and MRC-5 cell lines in the isolation of the mumps virus from clinical samples by means of the shell vial method. Methods—During an epidemic outbreak of parotiditis 48 clinical samples (saliva swabs and CSF) were studied. Two vials of the Vero, LLC-MK2, MDCK, MRC-5, and Hep-2 cell lines were inoculated with 0.2 ml of the samples by the shell vial assay. The vials were incubated at 36°C for two and five days. The vials were then fixed with acetone at -20°C for 10 minutes and stained by a monoclonal antibody against mumps virus by means of an indirect immunofluorescence assay. Results—The mumps virus was isolated from 36 samples. The Vero and LLC-MK2 cell lines showed a 100% isolation capacity, MDCK showed 77.7%, MRC-5 showed 44.4%, and Hep-2 showed 22.2%. The Vero and LLC-MK2 lines were significantly different to the other cell lines (p 5 infectious foci) were 94.4% for Vero, 97.2% for LLC-MK2, 5.5% for MDCK, 5.5% for Hep-2, and 0% for MRC-5. Conclusions—The Vero and LLC-MK2 cell lines are equally efficient at two and five days incubation for the isolation of the mumps virus from clinical samples, and the use of the shell vial method considerably shortens the time of aetiological diagnosis with higher specificity. Key Words: mumps virus • Vero cell line • LLC-MK2 cell line • MDCK cell line • Hep-2 cell line • MRC-5 cell line • isolation • shell vial PMID:11729211

  14. Comparison of Total RNA Isolation Methods for Analysis of Immune-Related microRNAs in Market Milks.

    Science.gov (United States)

    Oh, Sangnam; Park, Mi Ri; Son, Seok Jun; Kim, Younghoon

    2015-01-01

    Bovine milk provides essential nutrients, including immunologically important molecules, as the primary source of nutrition to newborns. Recent studies showed that RNAs from bovine milk contain immune-related microRNAs (miRNA) that regulate various immune systems. To evaluate the biological and immunological activity of miRNAs from milk products, isolation methods need to be established. Six methods for extracting total RNAs from bovine colostrums were adopted to evaluate the isolating efficiency and expression of miRNAs. Total RNA from milk was presented in formulation of small RNAs, rather than ribosomal RNAs. Column-combined phenol isolating methods showed high recovery of total RNAs, especially the commercial columns for biofluid samples, which demonstrated outstanding efficiency for recovering miRNAs. We also evaluated the quantity of five immune-related miRNAs (miR-93, miR-106a, miR-155, miR-181a, miR-451) in milk processed by temperature treatments including low temperature for long time (LTLT, 63℃ for 30 min)-, high temperature for short time (HTST, 75℃ for 15 s)-, and ultra heat treatment (UHT, 120-130℃ for 0.5-4 s). All targeted miRNAs had significantly reduced levels in processed milks compared to colostrum and raw mature milk. Interestingly, the amount of immune-related miRNAs from HTST milk was more resistant than those of LTLT and UHT milks. Our present study examined defined methods of RNA isolation and quantification of immune-specific miRNAs from small volumes of milk for use in further analysis.

  15. Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods.

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    Agnieszka E Laudy

    Full Text Available Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-β-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9, GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15, OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544 and OXA-10 (5 isolates with OXA-74 and one with OXA-142. The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide.

  16. A novel tertiary prep-HPLC method for the isolation of single amino acids for AMS-radiocarbon measurement.

    Science.gov (United States)

    Fernandes, Ricardo; Koudelka, Tomas; Tholey, Andreas; Dreves, Alexander

    2017-07-15

    AMS-radiocarbon measurements of amino acids can potentially provide more reliable radiocarbon dates than bulk collagen analysis. Nonetheless, the applicability of such an approach is often limited by the low-throughput of existing isolation methods and difficulties in determining the contamination introduced during the separation process. A novel tertiary prep-HPLC amino acid isolation method was developed that relies on the combustion of eluted material without requiring any additional chemical steps. Amino acid separation was carried out using a gradient mix of pure water and phosphoric acid with an acetonitrile step in-between runs to remove hydrophobic molecules from the separation column. The amount of contaminant carbon and its 14 C content were determined from two-point measurements of collagen samples of known 14 C content. The amount of foreign carbon due to the isolation process was estimated at 4±1μg and its 14 C content was 0.43±0.01 F 14 C. Radiocarbon values corrected for carbon contamination have only a minor increase in uncertainties. For Holocene samples, this corresponds to an added uncertainty typically smaller than 10 14 Cyears. The developed method can be added to routine AMS measurements without implying significant operational changes and offers a level of measurement uncertainty that is suitable for many archaeological, ecological, environmental, and biological applications. Copyright © 2017. Published by Elsevier B.V.

  17. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

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    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

  18. A screening method for the isolation of polyhydroxyalkanoate-producing purple non-sulfur photosynthetic bacteria from natural seawater

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    Mieko Higuchi-Takeuchi

    2016-09-01

    Full Text Available Polyhydroxyalkanoates (PHAs are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2 showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions.

  19. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

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    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  20. A Novel Antibiotic Mechanism of l-Cyclopropylalanine Blocking the Biosynthetic Pathway of Essential Amino Acid l-Leucine

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    Bingji Ma

    2017-12-01

    Full Text Available The unusual amino acid l-cyclopropylalanine was isolated from the mushroom Amanita virgineoides after detection in an anti-fungal screening test. l-Cyclopropylalanine was found to exhibit broad-spectrum inhibition against fungi and bacteria. The anti-fungal activity was found to be abolished in the presence of the amino acid l-leucine, but not any other amino acids, indicating that l-cyclopropylalanine may block the biosynthesis of the essential amino acid l-leucine, thereby inhibiting fungal and bacteria growth. Further biochemical studies found l-cyclopropylalanine indeed inhibits α-isopropylmalate synthase (α-IMPS, the enzyme that catalyzes the rate-limiting step in the biosynthetic pathway of l-leucine. Inhibition of essential l-leucine synthesis in fungal and bacteria organisms, a pathway absent in host organisms such as humans, may represent a novel antibiotic mechanism to counter the ever-increasing problem of drug resistance to existing antibiotics.

  1. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

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    Kenneth W. Witwer

    2013-05-01

    Full Text Available The emergence of publications on extracellular RNA (exRNA and extracellular vesicles (EV has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

  2. New method for laser driven ion acceleration with isolated, mass-limited targets

    International Nuclear Information System (INIS)

    Paasch-Colberg, T.; Sokollik, T.; Gorling, K.; Eichmann, U.; Steinke, S.; Schnuerer, M.; Nickles, P.V.; Andreev, A.; Sandner, W.

    2011-01-01

    A new technique to investigate laser driven ion acceleration with fully isolated, mass-limited glass spheres with a diameter down to 8μm is presented. A Paul trap was used to prepare a levitating glass sphere for the interaction with a laser pulse of relativistic intensity. Narrow-bandwidth energy spectra of protons and oxygen ions have been observed and were attributed to specific acceleration field dynamics in case of the spherical target geometry. A general limiting mechanism has been found that explains the experimentally observed ion energies for the mass-limited target.

  3. Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for omics analysis

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    Yunlong eLu

    2015-06-01

    Full Text Available The development of sperm cells from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput omics approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs, from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with sperm cells (SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative-cell nuclei (VN from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.

  4. Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for "omics" analysis.

    Science.gov (United States)

    Lu, Yunlong; Wei, Liqin; Wang, Tai

    2015-01-01

    The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput "omics" approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants.

  5. Flg22-Triggered Immunity Negatively Regulates Key BR Biosynthetic Genes.

    Science.gov (United States)

    Jiménez-Góngora, Tamara; Kim, Seong-Ki; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-01-01

    In plants, activation of growth and activation of immunity are opposing processes that define a trade-off. In the past few years, the growth-promoting hormones brassinosteroids (BR) have emerged as negative regulators of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), promoting growth at the expense of defense. The crosstalk between BR and PTI signaling was described as negative and unidirectional, since activation of PTI does not affect several analyzed steps in the BR signaling pathway. In this work, we describe that activation of PTI by the bacterial PAMP flg22 results in the reduced expression of BR biosynthetic genes. This effect does not require BR perception or signaling, and occurs within 15 min of flg22 treatment. Since the described PTI-induced repression of gene expression may result in a reduction in BR biosynthesis, the crosstalk between PTI and BR could actually be negative and bidirectional, a possibility that should be taken into account when considering the interaction between these two pathways.

  6. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

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    Mattanovich Diethard

    2008-07-01

    Full Text Available Abstract Background High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. Results Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1 is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP increases the riboflavin accumulation significantly. Conclusion The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

  7. Labelled precursors for biosynthetic studies on naphthylisoquinoline alkaloids

    International Nuclear Information System (INIS)

    Bringmann, Gerhard; Pokorny, Frank; Wenzel, Matthias; Wurm, Kathi; Schneider, Christoph

    1997-01-01

    The isotope labelled monocyclic ketones 5 and 8, postulated precursors to the presumably acetogenic naphthylisoquinoline alkaloids, have been synthesized for biogenetic experiments to Ancistrocladaceae and Dioncophyllaceae plants. Key step of the preparation of 1-(2'-[carbonyl- 14 C] acetyl-3',5'-dibenzyloxyphenyl-2-propanone ([ 14 C]-13 is the C-acetylation of the arylpropanone 10 with the mixed pivalic acetic anhydride ([ 14 C]-11). The resulting pyrylium salt [ 14 C]-12, which is stable and can be stored, is cleaved directly before the feeding experiment to give the diketone [ 14 C]-13 and deprotected to give the free phenolic target molecule [ 14 C]-5. This synthetic route is applicable also to the preparation of 1-(2'-[ 13 C 2 ]acetyl-3'hydroxyphenyl)-2-propanone ([ 13 C 2 ]-5) for biosynthetic experiments with NMR analysis. For the preparation of the oxygen-poorer 13 C-labelled diketone 1-(2'-[methyl- 13 C] acetyl-3'-hydr oxyphenyl)-2-propanone [ 13 C]-8, an 'indanone-route' has been elaborated. (Author)

  8. A reliable combination method to identification and typing of epidemic and endemic clones among clinical isolates of Acinetobacter baumannii.

    Science.gov (United States)

    Piran, Arezoo; Shahcheraghi, Fereshteh; Solgi, Hamid; Rohani, Mahdi; Badmasti, Farzad

    2017-10-01

    The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of bla OXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of bla OXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and bla OXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and bla OXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded bla OXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. An improved method for RNA isolation and cDNA library construction from immature seeds of Jatropha curcas L

    Directory of Open Access Journals (Sweden)

    Kaur Jatinder

    2010-05-01

    Full Text Available Abstract Background RNA quality and quantity is sometimes unsuitable for cDNA library construction, from plant seeds rich in oil, polysaccharides and other secondary metabolites. Seeds of jatropha (Jatropha curcas L. are rich in fatty acids/lipids, storage proteins, polysaccharides, and a number of other secondary metabolites that could either bind and/or co-precipitate with RNA, making it unsuitable for downstream applications. Existing RNA isolation methods and commercial kits often fail to deliver high-quality total RNA from immature jatropha seeds for poly(A+ RNA purification and cDNA synthesis. Findings A protocol has been developed for isolating good quality total RNA from immature jatropha seeds, whereby a combination of the CTAB based RNA extraction method and a silica column of a commercial plant RNA extraction kit is used. The extraction time was reduced from two days to about 3 hours and the RNA was suitable for poly(A+ RNA purification, cDNA synthesis, cDNA library construction, RT-PCR, and Northern hybridization. Based on sequence information from selected clones and amplified PCR product, the cDNA library seems to be a good source of full-length jatropha genes. The method was equally effective for isolating RNA from mustard and rice seeds. Conclusions This is a simple CTAB + silica column method to extract high quality RNA from oil rich immature jatropha seeds that is suitable for several downstream applications. This method takes less time for RNA extraction and is equally effective for other tissues where the quality and quantity of RNA is highly interfered by the presence of fatty acids, polysaccharides and polyphenols.

  10. Lectin-induced agglutination method of urinary exosomes isolation followed by mi-RNA analysis: Application for prostate cancer diagnostic.

    Science.gov (United States)

    Samsonov, Roman; Shtam, Tatiana; Burdakov, Vladimir; Glotov, Andrey; Tsyrlina, Evgenia; Berstein, Lev; Nosov, Alexander; Evtushenko, Vladimir; Filatov, Michael; Malek, Anastasia

    2016-01-01

    Prostate cancer is the most common cancer in men. Prostate-specific antigen has, however, insufficient diagnostic specificity. Novel complementary diagnostic approaches are greatly needed. MiRNAs are small regulatory RNAs which play an important role in tumorogenesis and are being investigated as a cancer biomarker. In addition to their intracellular regulatory functions, miRNAs are secreted into the extracellular space and can be found in various body fluids, including urine. The stability of extracellular miRNAs is defined by association with proteins, lipoprotein particles, and membrane vesicles. Among the known forms of miRNA packaging, tumour-derived exosome-enclosed miRNAs is thought to reflect the vital activity of cancer cells. The assessment of the exosomal fraction of urinary miRNA may present a new and highly specific method for prostate cancer diagnostics; however, this is challenged by the absence of reliable and inexpensive methods for isolation of exosomes. Prostate cancer (PC) cell lines and urine samples collected from 35 PC patients and 35 healthy donors were used in the study. Lectins, phytohemagglutinin, and concanavalin A were used to induce agglutination of exosomes. The efficiency of isolation process was evaluated by AFM and DLS assays. The protein content of isolated exosomes was analysed by western blotting. Exosomal RNA was assayed by automated electrophoresis and expression level of selected miRNAs was evaluated by RT-qPCR. The diagnostic potency of the urinary exosomal miRNA assessment was estimated by the ROC method. The formation of multi-vesicular agglutinates in urine can be induced by incubation with lectin at a final concentration of 2 mg/ml. These agglutinates contain urinary exosomes and may be pelleted by centrifugation with a relatively low G-force. The analysis of PC-related miRNA in urinary exosomes revealed significant up-regulation of miR-574-3p, miR-141-5p, and miR-21-5p associated with PC. Lectin-induced aggregation is a

  11. Isolation of hydroxytyrosol from olive leaves extract, radioiodination and investigation of bioaffinity using in vivo/in vitro methods

    Energy Technology Data Exchange (ETDEWEB)

    Ozkan, M.; Biber Muftuler, F.Z.; Kilcar, A. Yurt; Medine, E.I.; Unak, P. [Ege Univ., Izmir (Turkey). Dept. of Nuclear Applications

    2013-11-01

    It is known that medicinal plants like olive have biological activities due to their flavonoid content such as olueropein, tyrosol, hydroxytyrosol etc. In current study, hydroxytrosol (HT) which is one of the major phenolic compounds in olive, olive leaves and olive oil, was isolated after methanol extraction and purification of olive leaves which are grown in the northern Anatolia region of Turkey. The isolated HT was radiolabeled with {sup 131}I ({sup 131}I-HT) and the bioaffinity of this radiolabeled component of olive leaves extract was investigated by using in vivo/in vitro methods. It was found that HT could be radiolabeled with {sup 131}I in yields of 95.6 {+-} 4.4% (n = 8), and in vivo studies showed that {sup 131}I-HT is taken up by urinary bladder, stomach, small intestine, large intestine, breast and prostate. Significant incorporation of activity was observed in cell lines via in vitro studies. (orig.)

  12. Isolation of Candida dubliniensis for the first time in Cali, Colombia, and its identification with phenotyping methods.

    Science.gov (United States)

    Alvarez, María Inés; Suárez, Blanca Lynne; Caicedo, Luz Dary

    2009-01-01

    Candida dubliniensis is an emerging pathogenic yeast isolated mainly from the oral cavity of HIV-infected patients. The close phenotypic and genotypic relationship between C. albicans and C. dubliniensis has led to incorrectly identifying isolates of C. dubliniensis as C. albicans. The oral cavities of 107 diabetic patients were studied in Cali, Colombia, and 72 colonies of Candida, with shades of green on CHROMagar Candida culture media, were obtained. Various phenotypic tests were carried out, which included germ tube formation and production of chlamydospores on corn meal Agar. Additionally, growth studies were carried out at 42 degrees C and 45 degrees C and on Sabouraud agar with 6.5%, sodium chloride. Identification of C. dubliniensis with these tests was confirmed with API 20C Aux. We identified 65 and 7 colonies of C. albicans and C. dubliniensis, respectively. This is the first time that C. dubliniensis is identified with phenotypic methods in Colombia.

  13. [Explantation method of isolating a persistent tick-borne encephalitis virus from the organs of infected monkeys].

    Science.gov (United States)

    Levina, L S; Pogodina, V V

    1981-01-01

    The method of explantation was used to examine 63 organs from M. rhesus monkeys 92-783 days after intracerebral and subcutaneous inoculation with the Vasilchenko, Aina/1448 and 41/65 strains of tick-borne encephalitis virus. The optimal time for examination of the explants by tests of the hemagglutinating, cytopathogenic activity of the virus and its pathogenicity for mice was found to be the 15th day of cultivation. A comparative study of the properties of 3 isolates obtained from explants of the spleen, liver and subcortical cerebral ganglia 202 and 307 days after inoculation of monkeys was carried out. The isolates differed from the parental TBE virus strains by their capacity to form small plaques in PEKV cell cultures (pig embryo kidney cells in versen medium).

  14. A method of experimental rheumatoid arthritis induction using collagen type II isolated from chicken sternal cartilage.

    Science.gov (United States)

    Su, Zhaoliang; Shotorbani, Siamak Sandoghchian; Jiang, Xugan; Ma, Rui; Shen, Huiling; Kong, Fanzhi; Xu, Huaxi

    2013-07-01

    At present, collagen‑induced arthritis (CIA) is the best known and most extensively used model for the immunological and pathological characteristics of human rheumatoid arthritis (RA). This model is useful not only in aiding our understanding of the pathogenesis of this disease, but also in the development of new therapies. Bovine, porcine and human collagen has been used to induce CIA; however, response has been identified to vary between strains and injection conditions, and false positive results and reduced potency are common as a result of minor contaminants or deglycosylated protein. Therefore, in the present study, type II collagen (CII) was isolated and purified from chicken sternal cartilage and was found to successfully induce the RA model. Furthermore, T helper 17 (Th17) cells were observed to infiltrate the joint on day 45 following induction by CII. In vitro, expression of toll‑like receptor 2 (TLR2) increased in peritoneal macrophages stimulated by CII. In addition, blockage of TLR2 was identified to markedly decrease levels of TGF‑β and IL‑6 in the cell culture supernatant. The results indicate that CII isolated from chicken sternal cartilage may be recognized by TLR2 on macrophages, leading to TGF‑β and IL‑6 production and subsequent activation of Th17 cells which mediates CIA development.

  15. Comparison of two biochemical methods for identifying Corynebacterium pseudotuberculosis isolated from sheep and goats.

    Science.gov (United States)

    Huerta, Belén; Gómez-Gascón, Lidia; Vela, Ana I; Fernández-Garayzábal, José F; Casamayor, Almudena; Tarradas, Carmen; Maldonado, Alfonso

    2013-06-01

    The biochemical pattern of Cowan and Steel (BPCS) was compared with a commercial biochemical strip for the identification of Corynebacterium pseudotuberculosis isolated from small ruminants. On 16S rRNA gene sequencing, 40/78 coryneform isolates from the lymph nodes of sheep and goats with lesions resembling caseous lymphadenitis were identified as C. pseudotuberculosis. The sensitivities of the BPCS and the commercial biochemical strip relative to 16S rRNA sequencing were 80% and 85%, and their specificities were 92.1% and 94.7%, respectively; the level of agreement between the BPCS and the commercial biochemical strip was high (κ=0.82). Likelihood ratios for positive and negative results were 10.0 and 0.22 for the BPCS, and 16.0 and 0.16 for the commercial biochemical strip, respectively. These results indicate that the BPCS and the commercial biochemical strip are both useful for identifying C. pseudotuberculosis in veterinary microbiology laboratories. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Key roles of Arf small G proteins and biosynthetic trafficking for animal development.

    Science.gov (United States)

    Rodrigues, Francisco F; Harris, Tony J C

    2017-04-14

    Although biosynthetic trafficking can function constitutively, it also functions specifically for certain developmental processes. These processes require either a large increase to biosynthesis or the biosynthesis and targeted trafficking of specific players. We review the conserved molecular mechanisms that direct biosynthetic trafficking, and discuss how their genetic disruption affects animal development. Specifically, we consider Arf small G proteins, such as Arf1 and Sar1, and their coat effectors, COPI and COPII, and how these proteins promote biosynthetic trafficking for cleavage of the Drosophila embryo, the growth of neuronal dendrites and synapses, extracellular matrix secretion for bone development, lumen development in epithelial tubes, notochord and neural tube development, and ciliogenesis. Specific need for the biosynthetic trafficking system is also evident from conserved CrebA/Creb3-like transcription factors increasing the expression of secretory machinery during several of these developmental processes. Moreover, dysfunctional trafficking leads to a range of developmental syndromes.

  17. Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Naesby, Michael; Mortensen, Uffe Hasbro

    2013-01-01

    production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established...

  18. Heterologous stable expression of terpenoid biosynthetic genes using the moss Physcomitrella patens

    DEFF Research Database (Denmark)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin

    2014-01-01

    Heterologous and stable expression of genes encoding terpenoid biosynthetic enzymes in planta is an important tool for functional characterization and is an attractive alternative to expression in microbial hosts for biotechnological production. Despite improvements to the procedure, such as stre...

  19. [Comparison between conventional methods, ChromAgar Candida® and PCR method for the identification of Candida species in clinical isolates].

    Science.gov (United States)

    Estrada-Barraza, Deyanira; Dávalos Martínez, Arturo; Flores-Padilla, Luis; Mendoza-De Elias, Roberto; Sánchez-Vargas, Luis Octavio

    2011-01-01

    The increase in the incidence of yeast species causing fungemia in susceptible immunocompromised patients in the last two decades and the low sensitivity of conventional blood culture has led to the need to develop alternative approaches for the early detection and identification of causative species. The aim of this study was to compare the usefulness of molecular testing by the polymerase chain reaction (PCR) and conventional methods to identify clinical isolates of different species, using the ID32C ATB system (bioMérieux, France), chromogenic culture Chromagar Candida® (CHROMagar, France) and morphogenesis in corn meal agar. We studied 79 isolates, in which the most prevalent species using the system ID32C and PCR was C. albicans, followed by C. tropicalis, C. glabrata and C .krusei. PCR patterns obtained for the identification of clinical isolates were stable and consistent in the various independent studies and showed good reproducibility, concluding that PCR with species-specific primers that amplify genes ITS1 and ITS2 for rRNA or topoisomerase II primers is a very specific and sensitive method for the identification of C. glabrata, C. krusei, C. albicans, and with less specificity for C. tropicalis. Copyright © 2010 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  20. Comparison of Enzymatic Method Rapid Yeast Plus System with RFLP-PCR for Identification of Isolated Yeast from Vulvovaginal Candidiasis.

    Science.gov (United States)

    Hossein, Moallaei; Mirhendi, Seied Hossein; Brandão, João; Mirdashti, Reza; Rosado, Laura

    2011-09-01

    To compare two identification methods, i.e., restriction fragment length polymorphism (RFLP)-PCR analysis and enzymatic method Rapid TM Yeast Plus System to identify different species causing vulvovaginal candidiasis (VVC). Vaginal discharges of women who had attended the gynecology outpatient clinic of Mobini Hospital in Sabzevar, Iran were collected using cotton swabs and were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by germ-tube testing and Rapid TM Yeast Plus System (Remel USA). For molecular identification, the isolated DNA was amplified with ITS1 and ITS4 universal primers and PCR products digested with the enzyme HpaІІ followed by agarose gel electrophoresis. Epidemiological and clinical features of women with respect to identified species were also evaluated. Out of 231 subjects enrolled, 62 VVC cases were detected. The isolated species were identified as follows: Candida albicans, 24 (38.7%), C. glabrata, 15 (24.2%), C. kefyr, 13 (21.0%) C. krusei, 9 (14.5%), and Saccharomyces cerevisiae, 1 (1.6%) by RFLP-PCR method; whereas findings by Rapid TM Yeast Plus System were C. albicans, 24 (38.7%), C. glabrata, 5 (8%), C. kefyr, 11 (17.7%) C. krusei, 2 (3.2%), S. cerevisiae, 9 (14.5%), and C. tropicalis, 6 (9.6%) as well as other nonpathogenic yeasts, 4 (6.9%). Statistical comparison showed that there is no significant difference in identification of C. albicans by the two methods; although, in this study, it was not true about other species of yeasts. A correlation between clinical and laboratory findings is important as it enables us to administer an appropriate treatment on time.

  1. Comparative method of protein expression and isolation of EBV epitope in E.coli DH5α

    Science.gov (United States)

    Anyndita, Nadya V. M.; Dluha, Nurul; Himmah, Karimatul; Rifa'i, Muhaimin; Widodo

    2017-11-01

    Epstein-Barr Virus (EBV) or human herpes virus 4 (HHV-4) is a virus that infects human B cell and leads to nasopharyngeal carcinoma (NPC). The prevention of this disease remains unsuccessful since the vaccine has not been discovered. The objective of this study is to over-produce EBV gp350/220 epitope using several methods in E.coli DH5α. EBV epitope sequences were inserted into pMAL-p5x vector, then transformed into DH5α E.coli and over-produced using 0.3, 1 and 2 mM IPTG. Plasmid transformation was validated using AflIII restriction enzyme in 0.8% agarose. Periplasmic protein was isolated using 2 comparative methods and then analyzed using SDS-PAGE. Method A produced a protein band around 50 kDa and appeared only at transformant. Method B failed to isolate the protein, indicated by no protein band appearing. In addition, any variations in IPTG concentration didn't give a different result. Thus it can be concluded that even the lowest IPTG concentration is able to induce protein expression.

  2. Evaluation of different phenotypic methods for detection of amp c beta-lactamase producing bacteria in clinical isolates

    International Nuclear Information System (INIS)

    Hassan, A.; Usman, J.; Kalim, F.; Gill, M.M.; Khalid, A.; Iqbal, M.; Ingram, P.

    2013-01-01

    To compare the sensitivity and specificity of different phenotypic methods for detection of Amp C betalactamase producing bacteria. Study Design: Analytical study. Place and Duration of Study: Department of Microbiology, Army Medical College / National University of Sciences and Technology (NUST), Islamabad, Pakistan, from June 2010 to December 2010. Methodology: A total of 150 clinical isolates were screened for presence of Amp C beta-lactamase by using the cefoxitin disc. The confirmatory methods evaluated were inhibitor based assay (boronic acid), Amp C disc test and Amp C Etest. Three dimensional enzyme extract assay was used as the reference method for determining the sensitivity and specificity. Results: Among the total isolates tested, 62.8% bacteria showed the presence of Amp C beta-lactamase by standard three dimensional enzyme extract assay. Among the three methods compared, boronic acid disk test found out to be highly sensitive (88%) and specific (92%) for the detection of Amp C beta-lactamase producing bacteria. Conclusion: Detection of Amp C production is crucial in order to establish the antibiotic therapy and to attain the favourable clinical outcomes. Implementation of simple tests like boronic acid disk tests in the laboratories will help to alleviate the spread of Amp C beta-lactamase harboring organisms. (author)

  3. A nitrous acid biosynthetic pathway for diazo group formation in bacteria.

    Science.gov (United States)

    Sugai, Yoshinori; Katsuyama, Yohei; Ohnishi, Yasuo

    2016-02-01

    Although some diazo compounds have bioactivities of medicinal interest, little is known about diazo group formation in nature. Here we describe an unprecedented nitrous acid biosynthetic pathway responsible for the formation of a diazo group in the biosynthesis of the ortho-diazoquinone secondary metabolite cremeomycin in Streptomyces cremeus. This finding provides important insights into the biosynthetic pathways not only for diazo compounds but also for other naturally occurring compounds containing nitrogen-nitrogen bonds.

  4. Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips.

    Science.gov (United States)

    Unal, Meral; Palavan-Unsal, Narcin; Tufekci, M A

    2008-03-01

    The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

  5. Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plant-growth-promoting effect.

    Science.gov (United States)

    Passari, Ajit Kumar; Chandra, Preeti; Zothanpuia; Mishra, Vineet Kumar; Leo, Vincent Vineeth; Gupta, Vijai Kumar; Kumar, Brijesh; Singh, Bhim Pratap

    2016-10-01

    In the present study, fifteen endophytic actinobacterial isolates recovered from Solanum lycopersicum were studied for their antagonistic potential and plant-growth-promoting (PGP) traits. Among them, eight isolates showed significant antagonistic and PGP traits, identified by amplification of the 16S rRNA gene. Isolate number DBT204, identified as Streptomyces sp., showed multiple PGP traits tested in planta and improved a range of growth parameters in seedlings of chili (Capsicum annuum L.) and tomato (S. lycopersicum L.). Further, genes of indole acetic acid (iaaM) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) were successively amplified from five strains. Six antibiotics (trimethoprim, fluconazole, chloramphenicol, nalidixic acid, rifampicin and streptomycin) and two phytohormones [indole acetic acid (IAA) and kinetin (KI)] were detected and quantified in Streptomyces sp. strain DBT204 using UPLC-ESI-MS/MS. The study indicates the potential of these PGP strains for production of phytohormones and shows the presence of biosynthetic genes responsible for production of secondary metabolites. It is the first report showing production of phytohormones (IAA and KI) by endophytic actinobacteria having PGP and biosynthetic potential. We propose Streptomyces sp. strain DBT204 for inoculums production and development of biofertilizers for enhancing growth of chili and tomato seedlings. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  6. Systems and methods for enhancing isolation of high-temperature reactor containments

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, Per F.

    2017-09-26

    A high-temperature containment-isolation system for transferring heat from a nuclear reactor containment to a high-pressure heat exchanger is presented. The system uses a high-temperature, low-volatility liquid coolant such as a molten salt or a liquid metal, where the coolant flow path provides liquid free surfaces a short distance from the containment penetrations for the reactor hot-leg and the cold-leg, where these liquid free surfaces have a cover gas maintained at a nearly constant pressure and thus prevent high-pressures from being transmitted into the reactor containment, and where the reactor vessel is suspended within a reactor cavity with a plurality of refractory insulator blocks disposed between an actively cooled inner cavity liner and the reactor vessel.

  7. The Basalt Waste Isolation Project technical program evaluation process: A criteria-based method

    International Nuclear Information System (INIS)

    Babad, H.; Evans, C.; Wolfe, B.A.

    1982-01-01

    The need to objectively evaluate the progress being made by the Basalt Waste Isolation Project (BWIP) toward establishing the feasibility of siting a nuclear waste repository in basalt (NWRB) mandates a process for evaluating the technical work of the project. To assist BWIP management in the evaluation process, the Systems Department staff has developed a BWIP Technical Program Evaluation Process (TPEP). The basic process relates progress on project technical work to the BWIP Functional and System Performance Criteria as defined in National Waste Terminal Storage (NWTS) Criteria Documents. The benefits of the TPEP to BWIP and future plans for TPEP are discussed. During fiscal year (FY) 1982, TPEP will be further formalized and further applied to the review of BWIP technical activities

  8. Basalt Waste Isolation Project Technical Program Evaluation Process: a criteria-based method

    International Nuclear Information System (INIS)

    Babad, H.; Evans, G.C.; Wolfe, B.A.

    1982-01-01

    The need to objectively evaluate the progress being made by the Basalt Waste Isolation Project (BWIP) toward establishing the feasibility of siting a nuclear waste repository in basalt (NWRB) mandates a process for evaluating the technical work of the project. To assist BWIP management in the evaluation process, the Systems Department staff has developed a BWIP Technical Program Evaluation Process (TPEP). The basic process relates progress on project technical work to the SWIP Functional and System Performance Criteria as defined in National Waste Terminal Storage (MWTS) Criteria Documents. The benefits of the TPEP to BWIP and future plans for TPEP are discussed. During fiscal year (FY) 1982, TPEP wll be further formalized and further applied to the review of BWIP technical activities

  9. A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies

    Directory of Open Access Journals (Sweden)

    Lim Tit-Meng

    2010-10-01

    Full Text Available Abstract Background Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy. Results Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®. Conclusions The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic

  10. A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

    Directory of Open Access Journals (Sweden)

    Janosch Peter Heller

    2015-11-01

    Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

  11. Condensed summary of the systems prioritization method as a decision-aiding approach for the Waste Isolation Pilot Plant

    International Nuclear Information System (INIS)

    Boak, D.M.; Prindle, N.H.; Lincoln, R.

    1997-01-01

    In March 1994, the US Department of Energy Carlsbad Area Office (DOE/CAO) implemented a performance based decision-aiding method to assist in programmatic prioritization within the Waste Isolation Pilot Plant (WIPP) project. The prioritization was with respect to 40 CFR Part 191.13(a) and 40 CFR part 268.6. U.S. Environmental Protection Agency (EPA) requirements for long-term isolation of radioactive and hazardous wastes. The Systems Prioritization Method (SPM), was designed by Sandia National Laboratories to: (1) identify programmatic options (activities), their costs and durations; (2) analyze combinations of activities in terms of their predicted contribution to long-term performance of the WIPP disposal system; and (3) analyze cost, duration, and performance tradeoffs. SPM results were the basis for activities recommended to DOE/CAO in May 1995. SPM identified eight activities (less than 15% of the 58 proposed for consideration) predicted to be essential in addressing key regulatory issues. The SPM method proved useful for risk or performance-based prioritization in which options are interdependent and system behavior is nonlinear. 10 refs., 2 figs., 1 tab

  12. Isolation of UV-sensitive mutants of mouse L5178Y cells by a cell suspension spotting method

    International Nuclear Information System (INIS)

    Shiomi, T.; Hieda-Shiomi, N.; Sato, K.

    1982-01-01

    We have isolated 56 UV-sensitive mutant clones from a mouse L51 T/t line of L5178Y cells by a cell suspension spotting method. Five mutants have also been isolated from L51 T/t and L5178Y cells by the method reported by Thompson and coworkers. We divided the mutants into two groups, highly sensitive and moderately sensitive mutants, according to their sensitivity to UV irradiation. Fifty-eight mutants were highly sensitive and three were moderately sensitive to UV. The reconstruction experiments indicate that more than 90% of highly sensitive mutants were recovered by the cell suspension spotting method. Frequencies of recovered mutants highly sensitive to UV increased with increasing dose of mutagens. Recovered mutant frequency reached 10(-2) after treatment with 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (survival 0.2%). Eight UV-sensitive mutants were divided into four complementation groups. These mutants were 2-6 times more sensitive to UV than parental L51 T/t cells in terms of D37 (dose required to reduce survival to 37%). Four representative UV-sensitive mutants which are classified into different complementation groups were examined for their sensitivity to killing by UV, 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), X-rays, and MNNG. All four classes of mutants were found to be cross-sensitive to UV, 4NQO, and MMC, but not sensitive to X-rays and MNNG

  13. An inexpensive and fast method for infiltration coating of complex geometry matrices for ISOL production target applications

    International Nuclear Information System (INIS)

    Kawai, Y.; Alton, G.D.; Bilheux, J.-C.

    2005-01-01

    An inexpensive, fast, and close to universal infiltration coating technique has been developed for fabricating fast diffusion-release ISOL targets. Targets are fabricated by deposition of finely divided (∼1μm) compound materials in a paint-slurry onto highly permeable, complex structure reticulated-vitreous-carbon-foam (RVCF) matrices, followed by thermal heat treatment. In this article, we describe the coating method and present information on the physical integrity, uniformity of deposition, and matrix adherence of SiC, HfC and UC 2 targets, destined for on-line use as targets at the Holifield Radioactive Ion Beam Facility (HRIBF)

  14. Summary of the systems prioritization method (SPM) as a decision-aiding tool for the Waste Isolation Pilot Plant

    International Nuclear Information System (INIS)

    Boak, D.M.; Prindle, N.H.; Lincoln, R.; Mendenhall, F.; Weiner, R.; Bills, R.A.

    1996-01-01

    In March 1994, the Department of Energy Carlsbad Area Office (DOE/CAO) implemented a performance-based planning method to assist in programmatic prioritization within the Waste Isolation Pilot Plant (WIPP) project with respect to applicable Environmental Protection Agency (EPA) long-term performance requirements stated in 40 CFR 191.13(a) and 40 CFR 268.6. This method, the Systems Prioritization Method (SPM), was designed by Sandia National Laboratories (SNL) to: (1) identify programmatic options (activities) and their costs and durations; (2) analyze potential combinations of activities in terms of predicted contribution to long-term performance; and (3) analyze cost, duration, and performance tradeoffs. SPM results were the basis for recommendations to DOE/CAO in May 1995 for prioritization within the WIPP project. This paper presents a summary of the SPM implementation, key results, and lessons learned

  15. Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng. Mez

    Directory of Open Access Journals (Sweden)

    Leonardo Severo da Costa

    2016-06-01

    Full Text Available The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng. Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB-based and one sodium dodecyl sulfate (SDS-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT, in an oven at 40ºC (S40, and in a microwave oven (FMO. The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng. Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence amplification using PCR reactions and to get restriction fragments.

  16. Comparison of Molecular Typing Methods Useful for Detecting Clusters of Campylobacter jejuni and C. coli Isolates through Routine Surveillance

    Science.gov (United States)

    Taboada, Eduardo; Grant, Christopher C. R.; Blakeston, Connie; Pollari, Frank; Marshall, Barbara; Rahn, Kris; MacKinnon, Joanne; Daignault, Danielle; Pillai, Dylan; Ng, Lai-King

    2012-01-01

    Campylobacter spp. may be responsible for unreported outbreaks of food-borne disease. The detection of these outbreaks is made more difficult by the fact that appropriate methods for detecting clusters of Campylobacter have not been well defined. We have compared the characteristics of five molecular typing methods on Campylobacter jejuni and C. coli isolates obtained from human and nonhuman sources during sentinel site surveillance during a 3-year period. Comparative genomic fingerprinting (CGF) appears to be one of the optimal methods for the detection of clusters of cases, and it could be supplemented by the sequencing of the flaA gene short variable region (flaA SVR sequence typing), with or without subsequent multilocus sequence typing (MLST). Different methods may be optimal for uncovering different aspects of source attribution. Finally, the use of several different molecular typing or analysis methods for comparing individuals within a population reveals much more about that population than a single method. Similarly, comparing several different typing methods reveals a great deal about differences in how the methods group individuals within the population. PMID:22162562

  17. Evaluation of Novel Wet Chemistry Separation and Purification Methods to Facilitate Automation of Astatine-211 Isolation

    International Nuclear Information System (INIS)

    Wilbur, Daniel Scott

    2016-01-01

    % extracted; There was some indication that the PEG-Merrifield resins could be saturated (perhaps with Bi) resulting in lower capture percentages, but more studies need to be done to confirm that; A target dissolution chamber, designed and built at PNNL, works well with syringe pumps so it can be used in an automated system; Preliminary semi-automated 211 At isolation studies have been conducted with full-scale target dissolution and 211 At isolation using a PEG column on the Hamilton automated system gave low overall recoveries, but HNO 3 was used (rather than HCl) for loading the 211 At and flow rates were not optimized; Results obtained using PEG columns are high enough to warrant further development on a fully automated system; Results obtained also indicate that additional studies are warranted to evaluate other types of columns for 211 At separation from bismuth, which allow use of HNO 3 /HCl mixtures for loading and NaOH for eluting 211 At. Such a column could greatly simplify the overall isolation process and make it easier to automate.

  18. C25 steroid epimers produced by Penicillium janthinellum, a fungus isolated from fruits Melia azedarach

    Energy Technology Data Exchange (ETDEWEB)

    Marinho, Andrey M. do Rosario; Rodrigues-Filho, Edson; Ferreira, Antonio Gilberto [Sao Carlos Univ., SP (Brazil). Dept. de Quimica; Santos, Lourivaldo S. [Para Univ., Belem, PA (Brazil). Dept. de Quimica

    2005-11-15

    A plant-derived fungus, Penicillium janthinellum, obtained from Melia azedarach, produced ergosterol and ergosterol 5a,8a-peroxide along with a mixture of rare C25 steroid epimers. The C25 steroids, named neocyclocitrinols, shows exactly the same tetracyclic ring system present in cyclocitrinol, which was isolated from a sponge-derived Penicillium citrinum, with the same bicyclo [4:4:1] skeleton at A/B rings, but showing different side chains. The P. janthinellum was cultid over white corn and the three steroids were isolated by several silica gel based chromatographic procedures and identified by extensive NMR methods, mainly {sup 1}H - {sup 13}C correlations and {sup 1}H - {sup 1}H COSY and TOCSY. The biosynthetic origin of the cyclocitrinols is also discussed. (author)

  19. C25 steroid epimers produced by Penicillium janthinellum, a fungus isolated from fruits Melia azedarach

    International Nuclear Information System (INIS)

    Marinho, Andrey M. do Rosario; Rodrigues-Filho, Edson; Ferreira, Antonio Gilberto; Santos, Lourivaldo S.

    2005-01-01

    A plant-derived fungus, Penicillium janthinellum, obtained from Melia azedarach, produced ergosterol and ergosterol 5a,8a-peroxide along with a mixture of rare C25 steroid epimers. The C25 steroids, named neocyclocitrinols, shows exactly the same tetracyclic ring system present in cyclocitrinol, which was isolated from a sponge-derived Penicillium citrinum, with the same bicyclo [4:4:1] skeleton at A/B rings, but showing different side chains. The P. janthinellum was cultivated over white corn and the three steroids were isolated by several silica gel based chromatographic procedures and identified by extensive NMR methods, mainly 1 H - 13 C correlations and 1 H - 1 H COSY and TOCSY. The biosynthetic origin of the cyclocitrinols is also discussed. (author)

  20. Rapid, sensitive and cost effective method for isolation of viral DNA from feacal samples of dogs

    Directory of Open Access Journals (Sweden)

    Savi.

    2010-06-01

    Full Text Available A simple method for viral DNA extraction using chelex resin was developed. The method used was eco-friendly and cost effective compared to other methods such as phenol chloroform method which use health hazardous organic reagents. Further, a polymerase chain reaction (PCR based detection of canine parvovirus (CPV using primers from conserved region of VP2 gene was developed. To increase the sensitivity and specificity of reaction, nested PCR was designed. PCR reaction was optimized to amplify 747bp product of VP2 gene. The assay can be completed in few hours and doesn’t need hazardous chemicals. Thus, the sample preparation using chelating resin along with nested PCR seems to be a sensitive, specific and practical method for the detection of CPV in diarrhoeal feacal samples. [Vet. World 2010; 3(3.000: 105-106

  1. Isolation and detection of Listeria monocytogenes in poultry meat by standard culture methods and PCR

    Science.gov (United States)

    Kureljušić, J.; Rokvić, N.; Jezdimirović, N.; Kureljušić, B.; Pisinov, B.; Karabasil, N.

    2017-09-01

    Listeria is the genus of a bacteria found in soil and water and some animals, including poultry and cattle. It can be present in raw milk and food made from raw milk. It can also live in food processing plants and contaminate a variety of processed meats. Microscopically, Listeria species appear as small, Gram-positive rods, which are sometimes arranged in short chains. In direct smears, they can be coccoid, so they can be mistaken for streptococci. Longer cells can resemble corynebacteria. Flagella are produced at room temperature but not at 37°C. Haemolytic activity on blood agar has been used as a marker to distinguish Listeria monocytogenes among other Listeria species, but it is not an absolutely definitive criterion. Further biochemical characterization is necessary to distinguish between the different Listeria species. The objective of this study was to detect, isolate and identify Listeria monocytogenes from poultry meat. Within a period of six months from January to June 2017, a total of 15 samples were collected. Three samples were positive for the presence of Listeria monocytogenes. Biochemical and microbiological tests as well as PCR technique using specific primers were used to confirm L. Monocytogenes in the samples.

  2. Control Method Based on Demand Response Needs of Isolated Bus Regulation with Series-Resonant Converters for Residential Photovoltaic Systems

    Directory of Open Access Journals (Sweden)

    Shu-Huai Zhang

    2017-05-01

    Full Text Available Considering the effects of isolation and high efficiency, a series-resonant DC-DC converter (L-L-C type, with two inductors and a capacitor has been introduced into a residential photovoltaic (PV generation and storage system in this work, and a voltage gain curve upwarp drifting problem was found. In this paper, the reason of upwarp drifting in the voltage gain curve is given, and a new changing topological control method to solve the voltage regulation problem under light load conditions is proposed. Firstly, the ideal and actual first harmonic approximation (FHA models are given, and this drifting problem is ascribed to the multiple peaks of higher-order resonance between resonant tank and parasitic capacitors. Then the paper presents the pulse-frequency-modulation (PFM driver signals control method to translate the full-bridge LLC into a half-bridge LLC converter, and with this method the voltage gain could easily be reduced by half. Based on this method, the whole voltage and resonant current sharing control methods in on-line and off-line mode are proposed. The parameters design and optimization methods are also discussed in detail. Finally, a residential PV system platform based on the proposed parallel 7-kW full-bridge LLC converter is built to verify the proposed control method and theoretical analysis.

  3. Comparison of culture based methods for the isolation of Clostridium difficile from stool samples in a research setting.

    Science.gov (United States)

    Lister, Michelle; Stevenson, Emma; Heeg, Daniela; Minton, Nigel P; Kuehne, Sarah A

    2014-08-01

    Effective isolation of Clostridium difficile from stool samples is important in the research setting, especially where low numbers of spores/vegetative cells may be present within a sample. In this study, three protocols for stool culture were investigated to find a sensitive, cost effective and timely method of C. difficile isolation. For the initial enrichment step, the effectiveness of two different rich media, cycloserine-cefoxitin fructose broth (CCFB) and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared. For the comparison of four different, selective solid media; Cycloserine-cefoxitin fructose agar (CCFA), Cycloserine-cefoxitin egg yolk agar (CCEY), ChromID C. difficile and tryptone soy agar (TSA) with 5% sheep's blood with and without preceding broth enrichment were used. As a means to enable differentiation between C. difficile and other fecal flora, the effectiveness of the inclusion of a pH indictor (1% Neutral Red), was also evaluated. The data derived indicated that CCFB is more sensitive than CCMB-TAL, however, the latter had an improved recovery rate. A broth enrichment step had a reduced sensitivity over direct plating. ChromID C. difficile showed the best recovery rate whereas CCEY egg yolk agar was the most sensitive of the four. The addition of 1% Neutral Red did not show sufficient colour change when added to CCEY egg yolk agar to be used as a differential medium. For a low cost, timely and sensitive method of isolating C. difficile from stool samples we recommend direct plating onto CCEY egg yolk agar after heat shock. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies.

    Science.gov (United States)

    Waterman, Jenora T; Bird, David McK; Opperman, Charles H

    2006-03-01

    A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.

  5. Comparison of pathogen DNA isolation methods from large volumes of whole blood to improve molecular diagnosis of bloodstream infections.

    Directory of Open Access Journals (Sweden)

    Anne J M Loonen

    Full Text Available For patients suffering from bloodstream infections (BSI molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1-5 ml, the novel non-enzymatic procedure (Polaris, 1-5 ml, and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl. These methods were evaluated by processing blood spiked with 0-1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1-10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50-67% and EasyMAG (58-79%. For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70-75% (MolYsis 17-50% and TTE-EasyMAG 20-36%. The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction vs. 2 hours (MolYsis. In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.

  6. Plasma rich in growth factors promotes dermal fibroblast proliferation, migration and biosynthetic activity.

    Science.gov (United States)

    Anitua, E; Pino, A; Orive, G

    2016-11-02

    The use of plasma rich in growth factors (PRGF) has gained importance in many medical fields due to its regenerative potential. The aim of this study is to evaluate the effects of PRGF on primary skin fibroblasts assessing cell proliferation, migration and secretion of growth factors. The age of the patients from who PRGF was prepared was also studied to determine whether it influenced the outcomes. Human dermal fibroblasts were isolated from three healthy volunteers. Using PRGF-Endoret technology, PRGF was prepared from two groups of different ages (18-35 years and 50+ years). The effects of increasing concentration of PRGF (5%, 10% and 20%) on cell proliferation and migration was evaluated. Biosynthetic behaviour of cells was also analysed measuring vascular endothelial growth factor (VEGF), transforming growth factor b1 (TGFb1) and pro-collagen type I secreted levels with or without PRGF treatment. Mean platelet enrichment reached 2.4X and 2X in 18-35 and 50+ groups respectively. A dose-dependent response was observed in proliferation assays achieving the highest levels with 20% PRGF. Migration was also promoted in cells but not in a dose-dependent manner. Cell proliferation and migration outcomes obtained with PRGF (from both groups) were significantly higher compared to non-stimulated groups (pPRGF, however, with the exception of VEGF, no statistical significances were observed between the different age groups. Results from this study concluded that PRGF is safe and effective in stimulating skin regeneration by enhancing proliferation, migration and expression of pivotal bioactive molecules involved in wound healing and haemostasis.

  7. Regulation of the Omega-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

    Directory of Open Access Journals (Sweden)

    Marte Avranden Kjær

    Full Text Available Limited availability of the n-3 fatty acids EPA and DHA have led to an interest in better understanding of the n-3 biosynthetic pathway and its regulation. The biosynthesis of alpha-linolenic acid to EPA and DHA involves several complex reaction steps including desaturation-, elongation- and peroxisomal beta-oxidation enzymes. The aims of the present experiments were to gain more knowledge on how this biosynthesis is regulated over time by different doses and fatty acid combinations. Hepatocytes isolated from salmon were incubated with various levels and combinations of oleic acid, EPA and DHA. Oleic acid led to a higher expression of the Δ6 fatty acid desaturase (fad genes Δ6fad_a, Δ6fad_b, Δ6fad_c and the elongase genes elovl2 compared with cells cultured in medium enriched with DHA. Further, the study showed rhythmic variations in expression over time. Levels were reached where a further increase in specific fatty acids given to the cells not stimulated the conversion further. The gene expression of Δ6fad_a_and Δ6fad_b responded similar to fatty acid treatment, suggesting a co-regulation of these genes, whereas Δ5fad and Δ6fad_c showed a different regulation pattern. EPA and DHA induced different gene expression patterns, especially of Δ6fad_a. Addition of radiolabelled alpha-linolenic acid to the hepatocytes confirmed a higher degree of elongation and desaturation in cells treated with oleic acid compared to cells treated with DHA. This study suggests a complex regulation of the conversion process of n-3 fatty acids. Several factors, such as that the various gene copies are differently regulated, the gene expression show rhythmic variations and gene expression only affected to a certain level, determines when you get the maximum conversion of the beneficial n-3 fatty acids.

  8. Biosynthetic origin of acetic acid using SNIF-NMR; Determinacao da origem biossintetica de acido acetico atraves da tecnica 'Site Specific Natural Isotopic Fractionation Studied by Nuclear Magnetic Resonance (SNIF-NMR)'

    Energy Technology Data Exchange (ETDEWEB)

    Boffo, Elisangela Fabiana; Ferreira, Antonio Gilberto [Sao Carlos Univ., SP (Brazil). Dept. de Quimica

    2006-05-15

    The main purpose of this work is to describe the use of the technique Site-Specific Natural Isotopic Fractionation of hydrogen (SNIF-NMR), using {sup 2}H and {sup 1}H NMR spectroscopy, to investigate the biosynthetic origin of acetic acid in commercial samples of Brazilian vinegar. This method is based on the deuterium to hydrogen ratio at a specific position (methyl group) of acetic acitained by fermentation, through different biosynthetic mechanisms, which result in different isotopic ratios. We measured the isotopic ratio of vinegars obtained through C{sub 3}, C{sub 4}, and CAM biosynthetic mechanisms, blends of C{sub 3} and C{sub 4} (agrins) and synthetic acetic acid. (author)

  9. Development of a Method to Isolate Glutamic Acid from Foodstuffs for a Precise Determination of Their Stable Carbon Isotope Ratio.

    Science.gov (United States)

    Kobayashi, Kazuhiro; Tanaka, Masaharu; Yatsukawa, Yoichi; Tanabe, Soichi; Tanaka, Mitsuru; Ohkouchi, Naohiko

    2018-01-01

    Recent growing health awareness is leading to increasingly conscious decisions by consumers regarding the production and traceability of food. Stable isotopic compositions provide useful information for tracing the origin of foodstuffs and processes of food production. Plants exhibit different ratios of stable carbon isotopes (δ 13 C) because they utilized different photosynthetic (carbon fixation) pathways and grow in various environments. The origins of glutamic acid in foodstuffs can be differentiated on the basis of these photosynthetic characteristics. Here, we have developed a method to isolate glutamic acid in foodstuffs for determining the δ 13 C value by elemental analyzer-isotope-ratio mass spectrometry (EA/IRMS) without unintended isotopic fractionation. Briefly, following acid-hydrolysis, samples were defatted and passed through activated carbon and a cation-exchange column. Then, glutamic acid was isolated using preparative HPLC. This method is applicable to measuring, with a low standard deviation, the δ 13 C values of glutamic acid from foodstuffs derived from C3 and C4 plants and marine algae.

  10. Improved method for rapid and accurate isolation and identification of Streptococcus mutans and Streptococcus sobrinus from human plaque samples.

    Science.gov (United States)

    Villhauer, Alissa L; Lynch, David J; Drake, David R

    2017-08-01

    Mutans streptococci (MS), specifically Streptococcus mutans (SM) and Streptococcus sobrinus (SS), are bacterial species frequently targeted for investigation due to their role in the etiology of dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of these organisms in disease progression and the impact of the presence of either/both on a subject's caries experience. Of vital importance to the study of these organisms is an identification protocol that allows us to distinguish between the two species in an easy, accurate, and timely manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe, the need for a more rapid procedure for isolating and identifying high volumes of MS was recognized. We report here on the development of an accurate and rapid method for MS identification. Accuracy, ease of use, and material and time requirements for morphological differentiation on selective agar, biochemical tests, and various combinations of PCR primers were compared. The final protocol included preliminary identification based on colony morphology followed by PCR confirmation of species identification using primers targeting regions of the glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found to be highly accurate, more rapid than the previous methodology used, and easily learned. It resulted in more efficient use of both time and material resources. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Rural Latinos' mental wellbeing: a mixed-methods pilot study of family, environment and social isolation factors.

    Science.gov (United States)

    Stacciarini, Jeanne-Marie R; Smith, Rebekah; Garvan, Cynthia Wilson; Wiens, Brenda; Cottler, Linda B

    2015-05-01

    Upon immigration to the rural areas in the US, Latino families may experience cultural, geographic, linguistic and social isolation, which can detrimentally affect their wellbeing by acting as chronic stressors. Using a community engagement approach, this is a pilot mixed-method study with an embedded design using concurrent qualitative and quantitative data. The purpose of this study is to evaluate family and social environments in terms of protective factors and modifiable risks associated with mental well-being in Latino immigrants living in rural areas of Florida. Latino immigrant mother and adolescent dyads were interviewed by using in-depth ethnographic semistructured interviews and subsequent quantitative assessments, including a demographic questionnaire and three structured instruments: the Family Environment Scale Real Form, the SF-12v2™ Health Survey and the short version (eight items) of PROMIS Health Organization Social Isolation. This mixed-method pilot study highlighted how family, rural, and social environments can protect or impair wellbeing in rural Latino immigrant mother and adolescent dyads.

  12. A Modified Ficoll-Paque Gradient Method for Isolating Mononuclear Cells from the Peripheral and Umbilical Cord Blood of Humans for Biobanks and Clinical Laboratories.

    Science.gov (United States)

    Jia, Yanjuan; Xu, Hui; Li, Yonghong; Wei, Chaojun; Guo, Rui; Wang, Fang; Wu, Yu; Liu, Jing; Jia, Jing; Yan, Junwen; Qi, Xiaoming; Li, Yuanting; Gao, Xiaoling

    2018-04-01

    Although the Ficoll-Paque method is classically used to isolate peripheral blood mononuclear cells (PBMCs), modifications in this method are required for a more rapid and economic output for biobanks and clinical laboratories, particularly in developing countries. In this study, we addressed this issue by modifying the Ficoll-Paque method for the isolation of PBMCs or mononuclear cells from the peripheral and the umbilical cord blood of healthy and diseased (infected, anemic, and chronic obstructive pulmonary disease) adult individuals. In the modified method, we initiated the cell isolation process from the buffy coat layer, which appears in the interface between the plasma and sediments after centrifugation, instead of using the whole blood as described in the classic method. Although the PBMC yield by the modified method was about 12% less than in the classic method, the number of PBMCs isolated by the modified method was more than one million, which is enough for different research/diagnostic purposes, such as multi-omics detection. Assessment of cell viability and purity by hematology analyzer and trypan blue showed no significant difference between the viability and purity of the PBMCs isolated by these two methods in almost all groups, except samples from the infected and cord blood groups, where lower PBMC purity with higher granulocyte contamination were observed. In addition, at delayed processing time points, all parameters for the two methods were decreased in a time-dependent manner, especially at 8, 12, or 24 hours after the sample collection. In summary, the performance of PBMC isolation by the classic and modified methods mainly relies on the PBMC ratio in original samples. The modified method could be preferred for PBMC isolation because of its time and cost savings, especially for the biobanks and clinical laboratories in developing countries.

  13. Comparison of two silica-based extraction methods for DNA isolation from bones.

    Science.gov (United States)

    Rothe, Jessica; Nagy, Marion

    2016-09-01

    One of the most demanding DNA extractions is from bones and teeth due to the robustness of the material and the relatively low DNA content. The greatest challenge is due to the manifold nature of the material, which is defined by various factors, including age, storage, environmental conditions, and contamination with inhibitors. However, most published protocols do not distinguish between different types or qualities of bone material, but are described as being generally applicable. Our laboratory works with two different extraction methods based on silica membranes or the use of silica beads. We compared the amplification success of the two methods from bone samples with different qualities and in the presence of inhibitors. We found that the DNA extraction using the silica membrane method results an in higher DNA yield but also in a higher risk of co-extracting impurities, which can act as inhibitors. In contrast the silica beads method shows decreased co-extraction of inhibitors but also less DNA yield. Related to our own experiences it has to be considered that each bone material should be reviewed independently regarding the analysis and extraction method. Therefore, the most ambitious task is determining the quality of the bone material, which requires substantial experience. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production

    Directory of Open Access Journals (Sweden)

    Yang Haihua

    2010-01-01

    Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials. Results To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb was introduced into nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIKwas introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L-1, 4 -fold nikkomycin X and 210 mg L-1, 1.8-fold nikkomycin Z in the resulting exconjugants comparing with the parent strain (220 mg L-1 nikkomycin X and 120 mg L-1 nikkomycin Z. The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. Conclusion A high nikkomycins producing strain (1100 mg L-1 nikkomycins was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.

  15. Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources

    International Nuclear Information System (INIS)

    Nelson, D.L.; Ledbetter, S.A.; Corbo, L.; Victoria, M.F.; Ramirez-Solis, R.; Webster, T.D.; Ledbetter, D.H.; Caskey, C.T.

    1989-01-01

    Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. The authors report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. They demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. They also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions

  16. Method of isolation of traces of americium by using the +6 oxidation state properties

    International Nuclear Information System (INIS)

    Kwinta, Jean; Michel, Jean-Jacques

    1969-05-01

    The authors present a method to separate traces of americium from a solution containing fission products and actinides. This method comprises the following steps: firstly, the oxidation of americium at the +6 state by ammonium persulfate and carrying over of actinides and III and IV lanthanides by lanthanum fluoride; secondly, the reduction by hydrazine of the oxidized americium and carrying over of the reduced americium by lutetium fluoride; and thirdly, the americium-lutetium separation by selective extractions either with di 2 ethyl hexyl phosphoric acid, or by fractionated elution on an anionic resin column by a mixture of nitric acid and methanol [fr

  17. Enhanced computational methods for quantifying the effect of geographic and environmental isolation on genetic differentiation

    NARCIS (Netherlands)

    Botta, Filippo; Eriksen, Casper; Fontaine, Michael Christophe; Guillot, Gilles

    2015-01-01

    In a recent paper, Bradburd et al. (2013) proposed a model to quantify the relative effect ofgeographic and environmental distance on genetic differentiation. Here, we enhance this method in several ways. 1. We modify the covariance model so as to fit better with mainstream geostatistical models and

  18. A rapid cleanup method for the isolation and concentration of pyrrolizidine alkaloids in comfrey root.

    Science.gov (United States)

    Gray, Dean E; Porter, Andrew; O'Neill, Terry; Harris, Roger K; Rottinghaus, George E

    2004-01-01

    Preparations from comfrey (Symphytum officinale and S. x uplandicum) root and leaf contain varying levels of the hepatotoxic pyrrolizidine alkaloids (PAs). Reference compounds for comfrey are not commercially available, and there is currently no rapid extraction or analytical method capable of determining low levels in raw materials or as adulterants in commercially available extracts. A solid-phase extraction (SPE) method was developed using an Ergosil cleanup column that specifically binds the PAs. With this method, powdered comfrey root was extracted by sonication and shaking with basic chloroform. The extract was applied to the cleanup column under vacuum, washed with 2 mL acetone-chloroform (8 + 2, v/v) followed by 2 mL petroleum ether to remove excess chloroform. The column was dried under vacuum, and the PAs were eluted with 2 successive 1 mL aliquots methanol. Percent recoveries of the PAs following Ergosil SPE had an overall average of 96.8%, with RSD of 3.8% over a range of 1.0 to 25.0 g extracted in 100 mL. Average precision of the method (n = 3 over 4 extraction concentrations) gave an overall RSD of 6.0% for the 5 alkaloids, with a range of 0.8% (5 g in 100 mL) to 11.2% (25 g in 100 mL). Recovery optimization testing showed that 1.0 g comfrey root extracted in 100 mL yielded the greatest recovery (% dry weight) of the PAs, with an extraction efficiency and accuracy of 94.2%, and RSD of 1.7% (n = 9). The unique properties of the Ergosil cleanup column provide rapid sample cleanup, volume reduction, and concentration of PAs from comfrey extracts, and allow the eluant to be analyzed directly by traditional chromatographic methods.

  19. Modeling of Self-Excited Isolated Permanent Magnet Induction Generator Using Iterative Numerical Method

    Directory of Open Access Journals (Sweden)

    Mohamed Mostafa R.

    2016-01-01

    Full Text Available Self-Excited Permanent Magnet Induction Generator (PMIG is commonly used in wind energy generation systems. The difficulty of Self-Excited Permanent Magnet Induction Generator (SEPMIG modeling is the circuit parameters of the generator vary at each load conditions due to the a change in the frequency and stator voltage. The paper introduces a new modeling for SEPMIG using Gauss-sidle relaxation method. The SEPMIG characteristics using the proposed method are studied at different load conditions according to the wind speed variation, load impedance changes and different shunted capacitor values. The system modeling is investigated due to the magnetizing current variation, the efficiency variation, the power variation and power factor variation. The proposed modeling system satisfies high degree of simplicity and accuracy.

  20. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik

    2010-01-01

    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

  1. Method of targeted delivery of laser beam to isolated retinal rods by fiber optics.

    Science.gov (United States)

    Sim, Nigel; Bessarab, Dmitri; Jones, C Michael; Krivitsky, Leonid

    2011-11-01

    A method of controllable light delivery to retinal rod cells using an optical fiber is described. Photo-induced current of the living rod cells was measured with the suction electrode technique. The approach was tested with measurements relating the spatial distribution of the light intensity to photo-induced current. In addition, the ion current responses of rod cells to polarized light at two different orientation geometries of the cells were studied.

  2. A well-posed numerical method to track isolated conformal map singularities in Hele-Shaw flow

    International Nuclear Information System (INIS)

    Baker, G.; Siegel, M.; Tanveer, S.

    1995-01-01

    We present a new numerical method for calculating an evolving 2D Hele-Shaw interface when surface tension effects are neglected. In the case where the flow is directed from the less viscous fluid into the more viscous fluid, the motion of the interface is ill-posed; small deviations in the initial condition will produce significant changes in the ensuing motion. The situation is disastrous for numerical computation, as small roundoff errors can quickly lead to large inaccuracies in the computed solution. Our method of computation is most easily formulated using a conformal map from the fluid domain into a unit disk. The method relies on analytically continuing the initial data and equations of motion into the region exterior to the disk, where the evolution problem becomes well-posed. The equations are then numerically solved in the extended domain. The presence of singularities in the conformal map outside of the disk introduces specific structures along the fluid interface. Our method can explicitly track the location of isolated pole and branch point singularities, allowing us to draw connections between the development of interfacial patterns and the motion of singularities as they approach the unit disk. In particular, we are able to relate physical features such as finger shape, side-branch formation, and competition between fingers to the nature and location of the singularities. The usefulness of this method in studying the formation of topological singularities (self-intersections of the interface) is also pointed out. 47 refs., 10 figs., 1 tab

  3. Automated segmentation and isolation of touching cell nuclei in cytopathology smear images of pleural effusion using distance transform watershed method

    Science.gov (United States)

    Win, Khin Yadanar; Choomchuay, Somsak; Hamamoto, Kazuhiko

    2017-06-01

    The automated segmentation of cell nuclei is an essential stage in the quantitative image analysis of cell nuclei extracted from smear cytology images of pleural fluid. Cell nuclei can indicate cancer as the characteristics of cell nuclei are associated with cells proliferation and malignancy in term of size, shape and the stained color. Nevertheless, automatic nuclei segmentation has remained challenging due to the artifacts caused by slide preparation, nuclei heterogeneity such as the poor contrast, inconsistent stained color, the cells variation, and cells overlapping. In this paper, we proposed a watershed-based method that is capable to segment the nuclei of the variety of cells from cytology pleural fluid smear images. Firstly, the original image is preprocessed by converting into the grayscale image and enhancing by adjusting and equalizing the intensity using histogram equalization. Next, the cell nuclei are segmented using OTSU thresholding as the binary image. The undesirable artifacts are eliminated using morphological operations. Finally, the distance transform based watershed method is applied to isolate the touching and overlapping cell nuclei. The proposed method is tested with 25 Papanicolaou (Pap) stained pleural fluid images. The accuracy of our proposed method is 92%. The method is relatively simple, and the results are very promising.

  4. Evaluation of the Efficiency of Different Disruption Methods on Yeast Cell Wall Preparation for β-Glucan Isolation

    Directory of Open Access Journals (Sweden)

    Anna Bzducha-Wróbel

    2014-12-01

    Full Text Available Selected methods for yeast cell disruption were evaluated to establish their suitability for cell wall preparation in the process of β-glucan isolation. The effect of different disruption methods on contents of total saccharides, β-glucans and proteins in the produced cell walls preparations was analyzed. The degree of cell wall purification from intracellular components was established on the basis of the ratio of solubilised material. The investigated methods included: cell exposure to hot water (autoclaving, thermally-induced autolysis, homogenization in a bead mill, sonication and their combinations. Experimental systems were prepared in water (pH 5.0 and pH 7.0 and Tris-HCl buffer (pH 8.0. The Saccharomyces cerevisiae yeast cell wall preparations with the highest degree of cytosol component release and purification of β-glucans were produced by 30 min of cell homogenization with zirconium-glass beads (0.5 mm in diameter. This was confirmed by the highest ratio of solubilised material (approx. 64%–67%. The thus-produced preparations contained ca. 60% of total saccharides, 13%–14% of β(1,3/(1,6-glucans, and approx. 35% of crude proteins. Similar results were obtained after autolysis coupled with bead milling as well as with sonication, but the time required for these processes was more than 24 h. Homogenization in a bead mill could be valuable for general isolation procedures because allows one to eliminate the different autolytic activity of various yeast strains.

  5. Numerical Solutions for Nonlinear High Damping Rubber Bearing Isolators: Newmark's Method with Netwon-Raphson Iteration Revisited

    Science.gov (United States)

    Markou, A. A.; Manolis, G. D.

    2018-03-01

    Numerical methods for the solution of dynamical problems in engineering go back to 1950. The most famous and widely-used time stepping algorithm was developed by Newmark in 1959. In the present study, for the first time, the Newmark algorithm is developed for the case of the trilinear hysteretic model, a model that was used to describe the shear behaviour of high damping rubber bearings. This model is calibrated against free-vibration field tests implemented on a hybrid base isolated building, namely the Solarino project in Italy, as well as against laboratory experiments. A single-degree-of-freedom system is used to describe the behaviour of a low-rise building isolated with a hybrid system comprising high damping rubber bearings and low friction sliding bearings. The behaviour of the high damping rubber bearings is simulated by the trilinear hysteretic model, while the description of the behaviour of the low friction sliding bearings is modeled by a linear Coulomb friction model. In order to prove the effectiveness of the numerical method we compare the analytically solved trilinear hysteretic model calibrated from free-vibration field tests (Solarino project) against the same model solved with the Newmark method with Netwon-Raphson iteration. Almost perfect agreement is observed between the semi-analytical solution and the fully numerical solution with Newmark's time integration algorithm. This will allow for extension of the trilinear mechanical models to bidirectional horizontal motion, to time-varying vertical loads, to multi-degree-of-freedom-systems, as well to generalized models connected in parallel, where only numerical solutions are possible.

  6. Numerical Solutions for Nonlinear High Damping Rubber Bearing Isolators: Newmark’s Method with Netwon-Raphson Iteration Revisited

    Directory of Open Access Journals (Sweden)

    Markou A.A.

    2018-03-01

    Full Text Available Numerical methods for the solution of dynamical problems in engineering go back to 1950. The most famous and widely-used time stepping algorithm was developed by Newmark in 1959. In the present study, for the first time, the Newmark algorithm is developed for the case of the trilinear hysteretic model, a model that was used to describe the shear behaviour of high damping rubber bearings. This model is calibrated against free-vibration field tests implemented on a hybrid base isolated building, namely the Solarino project in Italy, as well as against laboratory experiments. A single-degree-of-freedom system is used to describe the behaviour of a low-rise building isolated with a hybrid system comprising high damping rubber bearings and low friction sliding bearings. The behaviour of the high damping rubber bearings is simulated by the trilinear hysteretic model, while the description of the behaviour of the low friction sliding bearings is modeled by a linear Coulomb friction model. In order to prove the effectiveness of the numerical method we compare the analytically solved trilinear hysteretic model calibrated from free-vibration field tests (Solarino project against the same model solved with the Newmark method with Netwon-Raphson iteration. Almost perfect agreement is observed between the semi-analytical solution and the fully numerical solution with Newmark’s time integration algorithm. This will allow for extension of the trilinear mechanical models to bidirectional horizontal motion, to time-varying vertical loads, to multi-degree-of-freedom-systems, as well to generalized models connected in parallel, where only numerical solutions are possible.

  7. Detection of Antimicrobial Compounds Isolated from Several Tropical Lentinus by Bioautographic Method

    Directory of Open Access Journals (Sweden)

    LISDAR I. SUDIRMAN

    2005-06-01

    Full Text Available The antimicrobial compounds extracted either from culture filtrates or mycelia of several tropical Lentinus species could be detected their existences and locations by bioautographic method. For this purpose, the crude extracts were deposited as spots on silica gel plates and developed in a n-butanol-acetic acid-water mixture (3:1:1. The dry silica gel plates were then seeded with Bacillus subtilis and incubated at 35 oC for one night. On these plates, the extracts were separated into several bioautographic spots or growth inhibition zones. In parallel, the spots were detected by viewing with chemical revelations or under ultraviolet radiations at 254 nm or 366 nm. On silica gel thin-layer chromatograms, the crude extracts of Lentinus were separated into several bioautographic spots; for the filtrate extracts of L. squarrosulus 55A into three spots (Rfs 0.75, 0.50, 0.17, the mycelial extracts of L. sajor-caju LSC8 into two spots (Rfs 0.77, 0.54, the mycelial extract of L. torulosus LU3 into two spots (Rfs 0.77, 0.48, the filtrate extracts of L. cladopus LC6 into one spot (Rf 0.76 but the mycelial extracts of this mushroom separated into two spots (Rfs 0.79, 0.54, the filtrate and mycelial extracts of L. cladopus LC4 into three spots respectively (Rfs 0.75, 0.61, 0.45 for the filtrate extract and Rfs 0.83, 0.73, 0.60 for mycelial extract. By this method, the active compounds were detected directly and it is a usual method for further work on the purification of the target compounds.

  8. Enhanced computational methods for quantifying the effect of geographic and environmental isolation on genetic differentiation

    DEFF Research Database (Denmark)

    Botta, Filippo; Eriksen, Casper; Fontaine, Michaël C.

    2015-01-01

    1. In a recent paper, Bradburd et al. (Evolution, 67, 2013, 3258) proposed a model to quantify the relative effect of geographic and environmental distance on genetic differentiation. Here, we enhance this method in several ways. 2. We modify the covariance model so as to fit better with mainstre...... available as an R package called sunder. It takes as input georeferenced allele counts at the individual or population level for co-dominant markers. Program homepage: http://www2.imm.dtu.dk/~gigu/Sunder/....

  9. Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee.

    Science.gov (United States)

    von Hertwig, Aline Morgan; Sant'Ana, Anderson S; Sartori, Daniele; da Silva, Josué José; Nascimento, Maristela S; Iamanaka, Beatriz Thie; Pelegrinelli Fungaro, Maria Helena; Taniwaki, Marta Hiromi

    2018-05-01

    Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B 2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate.

    Science.gov (United States)

    Manzo, Anthony J; Goushcha, Alexander O; Barabash, Yuri M; Kharkyanen, Valery N; Scott, Gary W

    2009-07-01

    Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I(exp), obeys a simple exponential law with the rate constant alphaI(exp) + k(rec), in which alpha is a parameter relating the light intensity, measured in mW/cm(2), to a corresponding theoretical rate in units of reciprocal seconds, and k(rec) is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the alpha parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer-Lambert-Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

  11. A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

    Science.gov (United States)

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

    2014-01-01

    The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

  12. Comparison of Some Extraction Methods for Isolation of Catechins and Caffeine from Turkish Green Tea

    Directory of Open Access Journals (Sweden)

    Ezgi DEMİR

    2015-07-01

    Full Text Available Effective extraction of anticancer and antioxidant principles from Turkish green tea were main purpose of this work. The pre-optimized experimental condition for liquid extraction was employed for comparative appraisal.  Not only extraction methods also nature of the green tea samples (fresh, dried or frozen and quantitative yields related to collection periods were investigated.  After extraction of the green tea with various techniques the extract was partitioned with chloroform to remove caffeine, after that the extract was partitioned with ethyl acetate to obtain catechin mixture. Quantification of individual catechins was carried out by HPLC and analysis results proved that epigallocatechin gallate (EGCG was main catechin specie present in all extracts. The results indicate that hot water extraction (at 80 0C provides higher catechin yield when compared to other methods. The highest extract yields were obtained with dried leaves collected in second collection period. The crude catechin mixture contains high amount of EGCG and might be used as raw material for production of plant remedies at industrial scale.

  13. A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone

    International Nuclear Information System (INIS)

    Venkatesan, Jayachandran; Ryu, BoMi; Thomas, Noel Vinay; Kim, Se Kwon; Qian Zhongji

    2011-01-01

    In the present study, hydroxyapatite (HAp) was isolated from Thunnus obesus bone using alkaline hydrolysis and thermal calcination methods. The obtained ceramic has been characterized by thermal gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), powder x-ray diffraction analysis (XRD), field-emission scanning electron microscopy, energy-dispersive x-ray analysis, transmission electron microscopy (TEM), selected area diffraction analysis, cytotoxic analysis and cell proliferation analysis. The results indicate that there are significant differences between the ceramics and T. obesus bone. FT-IR and TGA results affirmed that the collagen and organic moieties have been eliminated by both the proposed methods. XRD results were in agreement with JCPDS data. TEM and selective area diffraction images have signified that the thermal calcination method produces good crystallinity with dimensions 0.3-1.0 μm, whereas the alkaline hydrolysis method produces nanostructured HAp crystals with 17-71 nm length and 5-10 nm width. Biocompatibility of HAp crystals was evaluated by cytotoxicity and cell proliferation with human osteoblast-like cell MG-63.

  14. A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone

    Energy Technology Data Exchange (ETDEWEB)

    Venkatesan, Jayachandran; Ryu, BoMi; Thomas, Noel Vinay; Kim, Se Kwon [Department of Chemistry, Pukyong National University, Busan 608-737 (Korea, Republic of); Qian Zhongji, E-mail: sknkim@pknu.ac.kr [Marine Bioprocess Research Center, Pukyong National University, Busan 608-737 (Korea, Republic of)

    2011-06-15

    In the present study, hydroxyapatite (HAp) was isolated from Thunnus obesus bone using alkaline hydrolysis and thermal calcination methods. The obtained ceramic has been characterized by thermal gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), powder x-ray diffraction analysis (XRD), field-emission scanning electron microscopy, energy-dispersive x-ray analysis, transmission electron microscopy (TEM), selected area diffraction analysis, cytotoxic analysis and cell proliferation analysis. The results indicate that there are significant differences between the ceramics and T. obesus bone. FT-IR and TGA results affirmed that the collagen and organic moieties have been eliminated by both the proposed methods. XRD results were in agreement with JCPDS data. TEM and selective area diffraction images have signified that the thermal calcination method produces good crystallinity with dimensions 0.3-1.0 {mu}m, whereas the alkaline hydrolysis method produces nanostructured HAp crystals with 17-71 nm length and 5-10 nm width. Biocompatibility of HAp crystals was evaluated by cytotoxicity and cell proliferation with human osteoblast-like cell MG-63.

  15. Geometric spin frustration for isolated plaquettes of the lattices: An extended irreducible tensor operator method

    International Nuclear Information System (INIS)

    Wang Fan; Chen Zhida

    2006-01-01

    A new strategy to search for the good quantum numbers for the corner-sharing spin systems, as archetypal plaquettes of the lattices, was suggested for the first time in order to study on geometric spin frustration. The calculations on energy spectra by using the irreducible tensor operator method with the new strategy can be much reduced. As representative examples the energy spectra for the spin pentamer of the tetrahedron with a centered spin site and the spin heptamer of three corner-sharing equilateral-triangle were examined in order to confirm efficiency of the new strategy. Through our code, with automatically searching for the good quantum numbers, the projection operators S iz , S ix and S iy matrices in the ground state space for the spin heptamer were reliably constructed

  16. Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Wang

    Full Text Available Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+ cells. Moreover, CCSP(+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs in terminal bronchioles (TBs. We conclude that CCSP(+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

  17. Hydrodistillation-adsorption method for the isolation of water-soluble, non-soluble and high volatile compounds from plant materials.

    Science.gov (United States)

    Mastelić, J; Jerković, I; Blazević, I; Radonić, A; Krstulović, L

    2008-08-15

    Proposed method of hydrodistillation-adsorption (HDA) on activated carbon and hydrodistillation (HD) with solvent trap were compared for the isolation of water-soluble, non-soluble and high volatile compounds, such as acids, monoterpenes, isothiocyanates and others from carob (Certonia siliqua L.), rosemary (Rosmarinus officinalis L.) and rocket (Eruca sativa L.). Isolated volatiles were analyzed by GC and GC/MS. The main advantages of HDA method over ubiquitous HD method were higher yields of volatile compounds and their simultaneous separation in three fractions that enabled more detail analyses. This method is particularly suitable for the isolation and analysis of the plant volatiles with high amounts of water-soluble compounds. In distinction from previously published adsorption of remaining volatile compounds from distillation water on activated carbon, this method offers simultaneous hydrodistillation and adsorption in the same apparatus.

  18. Isolation and characterization of Ornithobacterium rhinotracheale in the commercial turkey, quail flocks and domestic pigeons by bacteriological and molecular methods

    Directory of Open Access Journals (Sweden)

    Banani, M.

    2011-12-01

    Full Text Available Ornithobacterium rhinotracheale (ORT is a respiratory pathogen which has been isolated throughout the world from numerous bird species. The present study was designed to isolate and characterize the ORT from domestic turkeys, quails and pigeons. For this purpose, 250 samples from each bird species (turkey, quail and pigeon with or without respiratory signs were tested by taking of tracheal swabs. In addition, respiratory tissue samples (tracheal and lung, from 250 slaughtered turkeys, 50 slaughtered quails and 100dead pigeons were also subjected to culture for ORT as tracheal swabs. Respiratory tissues were also tested for bacterial DNA by using polymerase chain reaction (PCR. In general, 30 isolates including 4 isolates from turkeys, 3 isolates from quails and 23 isolates from pigeons were identified as ORT by bacteriologicalmethod and then confirmed by PCR. Bacterial DNA was detected in 20%, 50% and 35% of respiratory tissues in turkeys, quails and pigeons respectively. Five ORT isolates from pigeon and all four isolates from turkey showed smaller colony size, while other isolates had larger colonies when cultured in blood agar. Fifty percent of the isolates with larger colony but none of the isolates with small colony size could agglutinate red blood cells (RBCs. All of the isolates were sensitive to danofloxacin and chloramphenicolwhile more than 90% of pigeon isolates were resistant to ampicillin. All of turkey and quail and 30% of pigeon isolates were resistant to tetracycline. Our ORT isolates showed high identity (98%- 100% insequence of 16S rRNA gene to related data in GeneBank.

  19. Comparison of different methods of RNA isolation for plum pox virus detection by reverse transcription-polymerase chain reaction.

    Science.gov (United States)

    Faggioli, F; Pasquini, G; Barba, M

    1998-09-01

    The diagnosis of plum pox virus (PPV) is still considered one of the most important aspects of the "sharka" problem. In fact, different studies demonstrated an uneven distribution of the virus in infected trees due to a high variability in virus concentration. These aspects complicate the PPV diagnosis. To date, biological, serological and molecular assays have been successively developed in order to obtain sensitive and efficient PPV detection techniques. In particular, the polymerase chain reaction (PCR) technique seems to be promising and can be considered the most sensitive and reliable one. Preparation of viral RNA is still a fundamental step in reverse transcription-PCR (RT-PCR) technique, especially when applied to large scale testing, i.e., for certification purposes. In order to find the most rapid and efficient procedure, we have compared three different procedures of extraction of viral RNA to be processed RT-PCR. Their common characteristics is their capacity to extract the RNA from a small amount of plant tissue without organic solvents in the extraction fluid. The procedures were as follows: an immuno-capture (IC) method using a specific antiserum, a silica-capture (SC) method using a non-specific matrix, and a simple and rapid RNA extraction (RE) method. They all were followed by one-tube RT-PCR. The obtained results show that all the three techniques allowed a successful amplification and detection of PPV in tested samples except the SC-PCR method which proved less effective. In fact, the IC-PCR and RE-PCR methods amplified and detected PPV in all isolates tested, while the SC-PCR method was able to reveal the presence of the virus in apricot and infected control samples only.

  20. Novel skeleton sesquiterpenoids isolated from guava leaves.

    Science.gov (United States)

    Ouyang, Wen; Zhu, Xiao-ai; Wang, Wei; Chen, Xue-Xiang; Chen, Yun-Jiao; Cao, Yong

    2016-01-01

    A chemical investigation of the plant Psidium guajava L., collected in Guangdong province, afforded two novel skeleton sesquiterpenoids 1 and 2. Compound 2 also known as isocaryolan-9-one was a new natural product. The structure of the novel compound 1 was determined as guavacid A by various spectroscopic methods. A possible biosynthetic pathway for 1 and 2 was proposed.

  1. Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

    Science.gov (United States)

    Horsman, Geoff P.; Chen, Yihua; Thorson, Jon S.; Shen, Ben

    2010-01-01

    Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence. PMID:20534556

  2. What is the evidence for the use of biologic or biosynthetic meshes in abdominal wall reconstruction?

    Science.gov (United States)

    Köckerling, F; Alam, N N; Antoniou, S A; Daniels, I R; Famiglietti, F; Fortelny, R H; Heiss, M M; Kallinowski, F; Kyle-Leinhase, I; Mayer, F; Miserez, M; Montgomery, A; Morales-Conde, S; Muysoms, F; Narang, S K; Petter-Puchner, A; Reinpold, W; Scheuerlein, H; Smietanski, M; Stechemesser, B; Strey, C; Woeste, G; Smart, N J

    2018-04-01

    Although many surgeons have adopted the use of biologic and biosynthetic meshes in complex abdominal wall hernia repair, others have questioned the use of these products. Criticism is addressed in several review articles on the poor standard of studies reporting on the use of biologic meshes for different abdominal wall repairs. The aim of this consensus review is to conduct an evidence-based analysis of the efficacy of biologic and biosynthetic meshes in predefined clinical situations. A European working group, "BioMesh Study Group", composed of invited surgeons with a special interest in surgical meshes, formulated key questions, and forwarded them for processing in subgroups. In January 2016, a workshop was held in Berlin where the findings were presented, discussed, and voted on for consensus. Findings were set out in writing by the subgroups followed by consensus being reached. For the review, 114 studies and background analyses were used. The cumulative data regarding biologic mesh under contaminated conditions do not support the claim that it is better than synthetic mesh. Biologic mesh use should be avoided when bridging is needed. In inguinal hernia repair biologic and biosynthetic meshes do not have a clear advantage over the synthetic meshes. For prevention of incisional or parastomal hernias, there is no evidence to support the use of biologic/biosynthetic meshes. In complex abdominal wall hernia repairs (incarcerated hernia, parastomal hernia, infected mesh, open abdomen, enterocutaneous fistula, and component separation technique), biologic and biosynthetic meshes do not provide a superior alternative to synthetic meshes. The routine use of biologic and biosynthetic meshes cannot be recommended.

  3. Antimicrobial activity evaluation and comparison of methods of susceptibility for Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter spp. isolates.

    Science.gov (United States)

    Rechenchoski, Daniele Zendrini; Dambrozio, Angélica Marim Lopes; Vivan, Ana Carolina Polano; Schuroff, Paulo Alfonso; Burgos, Tatiane das Neves; Pelisson, Marsileni; Perugini, Marcia Regina Eches; Vespero, Eliana Carolina

    The production of KPC (Klebsiella pneumoniae carbapenemase) is the major mechanism of resistance to carbapenem agents in enterobacterias. In this context, forty KPC-producing Enterobacter spp. clinical isolates were studied. It was evaluated the activity of antimicrobial agents: polymyxin B, tigecycline, ertapenem, imipenem and meropenem, and was performed a comparison of the methodologies used to determine the susceptibility: broth microdilution, Etest ® (bioMérieux), Vitek 2 ® automated system (bioMérieux) and disc diffusion. It was calculated the minimum inhibitory concentration (MIC) for each antimicrobial and polymyxin B showed the lowest concentrations for broth microdilution. Errors also were calculated among the techniques, tigecycline and ertapenem were the antibiotics with the largest and the lower number of discrepancies, respectively. Moreover, Vitek 2 ® automated system was the method most similar compared to the broth microdilution. Therefore, is important to evaluate the performance of new methods in comparison to the reference method, broth microdilution. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  4. Tiamulin activity against fastidious and nonfastidious veterinary and human bacterial isolates: initial development of in vitro susceptibility test methods.

    Science.gov (United States)

    Jones, Ronald N; Pfaller, Michael A; Rhomberg, Paul R; Walter, Donald H

    2002-02-01

    Tiamulin is a pleuromutilin derivative used in veterinary practice for the control and specific therapy of infections in swine. This report summarizes studies to establish standardized susceptibility testing methods, interpretive criteria, and reagent details for use in veterinary methods recently developed by the National Committee for Clinical Laboratory Standards (NCCLS) (standards M31-A and M37-A, NCCLS, Wayne, Pa., 1999). A total of 636 fastidious and nonfastidious animal and human pathogens were processed by using media and procedures described by the NCCLS. Tiamulin disk diffusion tests used a 30-microg disk concentration, and the proposed MIC breakpoints corresponding to levels achievable in animal target tissues (lung) were or =32 microg/ml for resistance. Correlate zone diameters for specific nonfastidious species were as follows: for Pasteurella multocida and staphylococci tested on Mueller-Hinton agar, susceptibility at > or =19 mm and resistance at or =16 mm and resistance at or =9 mm) was suggested for veterinary fastidious medium broth and enriched chocolate Mueller-Hinton agar. Absolute categorical agreement between NCCLS dilution and disk diffusion test results with these criteria ranged from 90.5 to 96.2%. Tiamulin susceptibility testing methods appear to be accurate in their categorical classification for indicated species, and their availability will allow immediate testing of animal isolates to guide therapy via appropriate levels of dosing and to monitor the development of resistance for agents in this unique class.

  5. New dereplication method applied to NMR-Based metabolomics on different fusarium species isolated from Rhizosphere of Senna spectabilis

    International Nuclear Information System (INIS)

    Selegato, Denise M.; Castro-Gamboa, Ian; Freire, Rafael T.; Tannús, Alberto

    2016-01-01

    The search for new sources of natural products steadily increased the use of bioinformatics tools that enabled efficient analysis of complex matrices. In this context, dereplication methods emerged as a fast way of identifying known compounds, accelerating the identification of bioactive chemotypes. Although 1 H NMR is widely used as an analytical technique, few studies have been reported using it as a dereplication tool, primarily because of the spectral complexity. This work aims to create a new computational method that analyses 1 H NMR data from Fusarium solani and F. oxysporum isolated from Senna spectabilis' srhizosphere through principal component analysis (PCA). The algorithm uses loading values to select important peaks that distinguish both species in PCA, allowing compound dereplication, even in highly similar profiles. As a result, the method, associated with other NMR experiments and information from an in-house Fusarium's metabolite library was able to distinguish different mycotoxins produced by both fungi, identifying fusaric acid and beauvericin for F. oxysporum and the depsipeptide HA23 from F. solani. (author)

  6. New dereplication method applied to NMR-Based metabolomics on different fusarium species isolated from Rhizosphere of Senna spectabilis

    Energy Technology Data Exchange (ETDEWEB)

    Selegato, Denise M.; Castro-Gamboa, Ian, E-mail: ian.castro@gmail.com [Universidade Estadual Paulista Júlio de Mesquita Filho (NuBBE/UNESP), Araraquara, SP (Brazil). Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais; Freire, Rafael T.; Tannús, Alberto [Universidade de São Paulo (CIERMag/USP), São Carlos, SP (Brazil). Centro de Imagens e Espectroscopia in Vivo por Ressonância Magnética

    2016-07-01

    The search for new sources of natural products steadily increased the use of bioinformatics tools that enabled efficient analysis of complex matrices. In this context, dereplication methods emerged as a fast way of identifying known compounds, accelerating the identification of bioactive chemotypes. Although {sup 1}H NMR is widely used as an analytical technique, few studies have been reported using it as a dereplication tool, primarily because of the spectral complexity. This work aims to create a new computational method that analyses {sup 1}H NMR data from Fusarium solani and F. oxysporum isolated from Senna spectabilis' srhizosphere through principal component analysis (PCA). The algorithm uses loading values to select important peaks that distinguish both species in PCA, allowing compound dereplication, even in highly similar profiles. As a result, the method, associated with other NMR experiments and information from an in-house Fusarium's metabolite library was able to distinguish different mycotoxins produced by both fungi, identifying fusaric acid and beauvericin for F. oxysporum and the depsipeptide HA23 from F. solani. (author)

  7. Lactobacillus genus identification isolated from gastrointestinal tract of chickens after bee products application using FISH and RTQ PCR methods

    Directory of Open Access Journals (Sweden)

    Miroslava Kačániová

    2013-05-01

    Full Text Available The general objective of this study was to examine the effect of bee products on the lactobacilli colonization of chickens. Bee products were administered to both feed mixtures in various amounts in addition to the control group. First experimental group was with propolis in feed mixture with the addition of 400 mg propolis per 1 kg of compound and second group was with pollen in feed mixture with the addition of 450 mg pollen per 1 kg of compound. In this experiment, quantitative counts of lactobacilli in ceca of 49-day-old chicken (Ross 308 using classical and FISH method were investigated. Counts of lactobacilli on MRS agar were monitored. To check the reliability of traditional methods of cultivation samples were evaluated by fluorescence in situ hybridization (FISH. Lactobacillus cells, isolated from gastrointestinal tract, were detected after hybridization of fluorescently labeled probe with bacterial cells. Counts of CFU of lactobacilli were compared in experimental and control treatments, respectively. The lowest count was detected in the control experimental group. The highest count was detected in the third experimental group where was 450 mg of pollen added to 1 kg of feed mixture. Using Real-time PCR method, we identified the species range of the genera Lactobacillus in the intestinal tract of broiler. Detected species from the genus Lactobacillus were L. crispatus, L. salivarius and L. acidophilus.

  8. The Improved Method for Isolation of Photochrome Trans-membrane Protein Bacteriorhodopsin from Purple Membranes of Halobacterium Halobacterium Halobium ET 1001

    OpenAIRE

    Oleg Mosin; Ignat Ignatov

    2015-01-01

    It was developed the improved method for isolation of photochrome trans-membraine protein bacteriorhodopsin (output – 5 mg from 100 g of wet biomass) capable to transform light energy to electrochemical energy of generated protons H+ and АТP. The protein was isolated from purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001 by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weigh...

  9. Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach

    Science.gov (United States)

    2012-01-01

    Background Profiling sperm DNA present on vaginal swabs taken from rape victims often contributes to identifying and incarcerating rapists. Large amounts of the victim’s epithelial cells contaminate the sperm present on swabs, however, and complicate this process. The standard method for obtaining relatively pure sperm DNA from a vaginal swab is to digest the epithelial cells with Proteinase K in order to solubilize the victim’s DNA, and to then physically separate the soluble DNA from the intact sperm by pelleting the sperm, removing the victim’s fraction, and repeatedly washing the sperm pellet. An alternative approach that does not require washing steps is to digest with Proteinase K, pellet the sperm, remove the victim’s fraction, and then digest the residual victim’s DNA with a nuclease. Methods The nuclease approach has been commercialized in a product, the Erase Sperm Isolation Kit (PTC Labs, Columbia, MO, USA), and five crime laboratories have tested it on semen-spiked female buccal swabs in a direct comparison with their standard methods. Comparisons have also been performed on timed post-coital vaginal swabs and evidence collected from sexual assault cases. Results For the semen-spiked buccal swabs, Erase outperformed the standard methods in all five laboratories and in most cases was able to provide a clean male profile from buccal swabs spiked with only 1,500 sperm. The vaginal swabs taken after consensual sex and the evidence collected from rape victims showed a similar pattern of Erase providing superior profiles. Conclusions In all samples tested, STR profiles of the male DNA fractions obtained with Erase were as good as or better than those obtained using the standard methods. PMID:23211019

  10. Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

    Science.gov (United States)

    Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

    2010-11-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

  11. SANDPUMA: ensemble predictions of nonribosomal peptide chemistry reveal biosynthetic diversity across Actinobacteria.

    Science.gov (United States)

    Chevrette, Marc G; Aicheler, Fabian; Kohlbacher, Oliver; Currie, Cameron R; Medema, Marnix H

    2017-10-15

    Nonribosomally synthesized peptides (NRPs) are natural products with widespread applications in medicine and biotechnology. Many algorithms have been developed to predict the substrate specificities of nonribosomal peptide synthetase adenylation (A) domains from DNA sequences, which enables prioritization and dereplication, and integration with other data types in discovery efforts. However, insufficient training data and a lack of clarity regarding prediction quality have impeded optimal use. Here, we introduce prediCAT, a new phylogenetics-inspired algorithm, which quantitatively estimates the degree of predictability of each A-domain. We then systematically benchmarked all algorithms on a newly gathered, independent test set of 434 A-domain sequences, showing that active-site-motif-based algorithms outperform whole-domain-based methods. Subsequently, we developed SANDPUMA, a powerful ensemble algorithm, based on newly trained versions of all high-performing algorithms, which significantly outperforms individual methods. Finally, we deployed SANDPUMA in a systematic investigation of 7635 Actinobacteria genomes, suggesting that NRP chemical diversity is much higher than previously estimated. SANDPUMA has been integrated into the widely used antiSMASH biosynthetic gene cluster analysis pipeline and is also available as an open-source, standalone tool. SANDPUMA is freely available at https://bitbucket.org/chevrm/sandpuma and as a docker image at https://hub.docker.com/r/chevrm/sandpuma/ under the GNU Public License 3 (GPL3). chevrette@wisc.edu or marnix.medema@wur.nl. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  12. Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases

    NARCIS (Netherlands)

    Duitman, Erwin H.; Wyczawski, Dobek; Boven, Ludolf G.; Venema, Gerard; Kuipers, Oscar P.; Hamoen, Leendert W.

    Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates

  13. Isolation of αL I domain mutants mediating firm cell adhesion using a novel flow-based sorting method.

    Science.gov (United States)

    Pepper, Lauren R; Parthasarathy, Ranganath; Robbins, Gregory P; Dang, Nicholas N; Hammer, Daniel A; Boder, Eric T

    2013-08-01

    The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.

  14. Influence of Bovine Whey Protein Concentrate and Hydrolysate Preparation Methods on Motility in the Isolated Rat Distal Colon

    Science.gov (United States)

    Dalziel, Julie E.; Anderson, Rachel C.; Bassett, Shalome A.; Lloyd-West, Catherine M.; Haggarty, Neill W.; Roy, Nicole C.

    2016-01-01

    Whey protein concentrate (WPC) and hydrolysate (WPH) are protein ingredients used in sports, medical and pediatric formulations. Concentration and hydrolysis methods vary for whey sourced from cheese and casein co-products. The purpose of this research was to investigate the influence of whey processing methods on in vitro gastrointestinal (GI) health indicators for colonic motility, epithelial barrier integrity and immune modulation. WPCs from casein or cheese processing and WPH (11% or 19% degree of hydrolysis, DH) were compared for their effects on motility in a 1 cm section of isolated rat distal colon in an oxygenated tissue bath. Results showed that WPC decreased motility irrespective of whether it was a by-product of lactic acid or mineral acid casein production, or from cheese production. This indicated that regardless of the preparation methodology, the whey protein contained components that modulate aspects of motility within the distal colon. WPH (11% DH) increased contractile frequency by 27% in a delayed manner and WPH (19% DH) had an immediate effect on contractile properties, increasing tension by 65% and frequency by 131%. Increased motility was associated with increased hydrolysis that may be attributed to the abundance of bioactive peptides. Increased frequency of contractions by WPH (19% DH) was inhibited (by 44%) by naloxone, implicating a potential involvement of opioid receptors in modulation of motility. Trans-epithelial electrical resistance and cytokine expression assays revealed that the WPC proteins studied did not alter intestinal barrier integrity or elicit any discernible immune response. PMID:27983629

  15. A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

    Directory of Open Access Journals (Sweden)

    Lorena Vázquez-Iglesias

    2017-06-01

    Full Text Available Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

  16. Radioactive beams produced by the ISOL method: development for laser ionization and for surface ionization; Faisceaux exotiques par methode ISOL: developpements pour l'ionisation par laser et l'ionisation de surface

    Energy Technology Data Exchange (ETDEWEB)

    Hosni, Faouzi

    2004-10-01

    The works were carried out in the framework of the research program PARRNe (production of radioactive neutron-rich nuclei). This program aims to determine optimal conditions to produce intense beams of neutron-rich isotopes. This thesis treats multiple technical aspects related to the production of separate radioactive isotopes in line (ISOL). It deals mainly with the development of the target-source unit which is the key element for projects such as SPIRAL-2 or EURISOL.The first part presents the various methods using fission as production mode and compares them: fission induced by thermal neutrons, induced by fast neutrons and photofission. The experiment carried out at CERN validated the interest of the photofission as a promising production mode of radioactive ions. That is why the institute of nuclear physics of Orsay decided to build a linear electron accelerator at the Tandem d'Orsay (ALTO).The second part of this thesis deals with the development of uranium targets. The X-rays diffraction and Scanning Electron Microscopy have been used as analysis techniques. They allowed to determine the chemical and structural characteristics of uranium carbide targets as function of various heating temperatures. After the production, the process of ionization has been studied. Two types of ion source have been worked out: the first one is a surface ion source and the second one is a source based on resonant ionization by laser. These two types of sources will be used for the ALTO project. (author)

  17. Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

    Directory of Open Access Journals (Sweden)

    Maria Inês Moura Freixo

    2004-02-01

    Full Text Available Mycobacterium tuberculosis strains resistant to streptomycin (SM, isoniazid (INH, and/or rifampin (RIF as determined by the conventional Löwenstein-Jensen proportion method (LJPM were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB. Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63% for SM and none for INH when isolates were re-tested but worsened for RIF (30%. Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.

  18. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    Full Text Available Herba epimedii (Epimedium, a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1 from Epimedium sagittatum (Sieb. Et Zucc. Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR and anthocyanidin synthase (ANS. In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

  19. A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

    Science.gov (United States)

    Huang, Wenjun; Sun, Wei; Lv, Haiyan; Luo, Ming; Zeng, Shaohua; Pattanaik, Sitakanta; Yuan, Ling; Wang, Ying

    2013-01-01

    Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

  20. Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, María Angeles; Sabater-Jara, Ana Belén

    2018-04-21

    Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight -1 ) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota

  1. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis.

    Science.gov (United States)

    Ohyama, Kiyoshi; Suzuki, Masashi; Kikuchi, Jun; Saito, Kazuki; Muranaka, Toshiya

    2009-01-20

    The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-(13)C(2)H(3)]mevalonate with Arabidopsis seedlings. Applying (13)C-{(1)H}{(2)H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol "the lanosterol pathway." LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as beta-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites.

  2. Characterization of the biosynthetic gene cluster for cryptic phthoxazolin A in Streptomyces avermitilis.

    Directory of Open Access Journals (Sweden)

    Dian Anggraini Suroto

    Full Text Available Phthoxazolin A, an oxazole-containing polyketide, has a broad spectrum of anti-oomycete activity and herbicidal activity. We recently identified phthoxazolin A as a cryptic metabolite of Streptomyces avermitilis that produces the important anthelmintic agent avermectin. Even though genome data of S. avermitilis is publicly available, no plausible biosynthetic gene cluster for phthoxazolin A is apparent in the sequence data. Here, we identified and characterized the phthoxazolin A (ptx biosynthetic gene cluster through genome sequencing, comparative genomic analysis, and gene disruption. Sequence analysis uncovered that the putative ptx biosynthetic genes are laid on an extra genomic region that is not found in the public database, and 8 open reading frames in the extra genomic region could be assigned roles in the biosynthesis of the oxazole ring, triene polyketide and carbamoyl moieties. Disruption of the ptxA gene encoding a discrete acyltransferase resulted in a complete loss of phthoxazolin A production, confirming that the trans-AT type I PKS system is responsible for the phthoxazolin A biosynthesis. Based on the predicted functional domains in the ptx assembly line, we propose the biosynthetic pathway of phthoxazolin A.

  3. Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

    NARCIS (Netherlands)

    Elfahmi, [No Value

    2006-01-01

    In this thesis phytochemical and biosynthetic studies of lignans are described. The focus is on the Indonesian medicinal plants Phyllanthus niruri and Piper cubeba and on two Linum species, Linum flavum and L. leonii, native to European countries. Both Indonesian plants are used in jamu. Jamu is the

  4. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol

    NARCIS (Netherlands)

    Cerneus, D. P.; Ueffing, E.; Posthuma, G.; Strous, G. J.; van der Ende, A.

    1993-01-01

    Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this

  5. Distribution of secondary metabolite biosynthetic gene clusters in 343 Fusarium genomes

    Science.gov (United States)

    Fusarium consists of over 200 phylogenetically distinct species, many of which cause important crop diseases and/or produce mycotoxins and other secondary metabolites (SMs). Some fusaria also cause opportunistic infections in humans and other animals. To investigate the distribution of biosynthetic ...

  6. Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

    NARCIS (Netherlands)

    El Khattabi, Mohamed; van Roosmalen, Maarten L.; Jager, Dennis; Metselaar, Heidi; Permentier, Hjalmar; Leenhouts, Kees; Broos, Jaap

    2008-01-01

    Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorporation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is

  7. Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters

    NARCIS (Netherlands)

    Cimermancic, P.; Medema, Marnix; Claesen, J.; Kurika, K.; Wieland Brown, L.C.; Mavrommatis, K.; Pati, A.; Godfrey, P.A.; Koehrsen, M.; Clardy, J.; Birren, B. W.; Takano, Eriko; Sali, A.; Linington, R.G.; Fischbach, M.A.

    2014-01-01

    Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the

  8. New insights into the organization and regulation of trichothecene biosynthetic genes in Trichoderma

    Science.gov (United States)

    Collectively, species of the genus Trichoderma can produce numerous structurally diverse secondary metabolites (SM). This ability is conferred by the presence of SM biosynthetic gene clusters in their genomes. Species of Trichoderma in the Brevicompactum clade are able to produce trichothecenes, a f...

  9. Characterization and engineering of thermophilic aldolases : synthesizing nitrogen-heterocycles in biosynthetic routes

    NARCIS (Netherlands)

    Wolterink-van Loo, S.

    2009-01-01

    Aldolases are enzymes that catalyze reactions in both degradation and biosynthetic pathways in vivo and have been discovered in all domains of life. they. An interesting property of aldolases is that they can synthesize carbon-carbon bonds, generating a new stereogenic centre. As enzymes are

  10. Neurosteroid biosynthetic pathway changes in substantia nigra and caudate nucleus in Parkinson's disease

    NARCIS (Netherlands)

    Luchetti, Sabina; Bossers, Koen; Frajese, Giovanni Vanni; Swaab, Dick F.

    2010-01-01

    There is emerging evidence from animal studies for a neuroprotective role of sex steroids in neurodegenerative diseases, but studies in human brain are lacking. We have carried out an extensive study of the neurosteroid biosynthetic pathways in substantia nigra (SN), caudate nucleus (CN) and putamen

  11. The oxalic acid biosynthetic activity of Burkholderia mallei is encoded by a single locus

    Science.gov (United States)

    Although it is known that oxalic acid provides a selective advantage to the secreting microbe, our understanding of how this acid is biosynthesized remains incomplete. This study reports the identification, cloning, and partial characterization of the oxalic acid biosynthetic enzyme from the animal ...

  12. An Integrated Metabolomic and Genomic Mining Workflow to Uncover the Biosynthetic Potential of Bacteria

    DEFF Research Database (Denmark)

    Månsson, Maria; Vynne, Nikolaj Grønnegaard; Klitgaard, Andreas

    2016-01-01

    Microorganisms are a rich source of bioactives; however, chemical identification is a major bottleneck. Strategies that can prioritize the most prolific microbial strains and novel compounds are of great interest. Here, we present an integrated approach to evaluate the biosynthetic richness in ba...

  13. Biochemical and physicochemical analysis of fish protein isolate recovered from red snapper (Lutjanus sp.) by-product using isoelectric solubilization/precipitation method

    Science.gov (United States)

    Pramono, H.; Pujiastuti, D. Y.; Sahidu, A. M.

    2018-04-01

    The effect of acid- and alkali-process on biochemical and physicochemical characteristics of fish protein isolate from red snapper (Lutjanus sp) by-product was evaluated. Protein recovered by alkali process (16.79%) was higher compared to acid process (13.75%). Reduction of lipid content and total volatile basic nitrogen (TVB-N) exhibited in both treatments indicated both process improved fish protein isolate recovered from red snapper by-product. In addition, the increasing of water holding capacity and oil binding capacity were observed. However, high peroxide value of fish protein isolate was showed in both treatment. This finding indicated that acid and alkali process can be used as a useful method to recover proteins from red snapper by-product. Alkali process gave a protein isolate with better overall quality compared to acid process.

  14. Automatic speech recognition (zero crossing method). Automatic recognition of isolated vowels; Reconnaissance automatique de la parole (methode des passages par zero). Reconnaissance automatique de voyelles isolees

    Energy Technology Data Exchange (ETDEWEB)

    Dupeyrat, Benoit

    1975-06-10

    This note describes a recognition method of isolated vowels, using a preprocessing of the vocal signal. The processing extracts the extrema of the vocal signal and the interval time separating them (Zero crossing distances of the first derivative of the signal). The recognition of vowels uses normalized histograms of the values of these intervals. The program determines a distance between the histogram of the sound to be recognized and histograms models built during a learning phase. The results processed on real time by a minicomputer, are relatively independent of the speaker, the fundamental frequency being not allowed to vary too much (i.e. speakers of the same sex). (author) [French] Cette note decrit une methode de reconnaissance automatique de voyelles isolees basee sur un pretraitement particulier du signal vocal. Ce pretraitement consiste a extraire les extrema du signal vocal et les intervalles de temps les separant (distances entre passages par zero de la derivee du signal). La reconnaissance des voyelles est faite en utilisant des histogrammes normalises des valeurs de ces interval les. Le programme de reconnaissance utilise une distance entre l'histogramme du son a reconnaitre et des histogrammes modeles provenant d'un apprentissage. Les resultats obtenus en temps reels sur un minicalculateur, sont assez independants du locuteur, pourvu que la frequence fondamentale de la voix ne varie pas trop (locuteurs de meme sexe). (auteur)

  15. Bioenergetic coupling between membrane transport systems and biosynthetic pathways essential for cell cycle progression

    International Nuclear Information System (INIS)

    Leister, K.J.; Cutry, A.F.; Wenner, C.E.

    1986-01-01

    Recently, it has been shown that there exists a point in the cell cycle (approximately 2 h prior to S phase entry) when (Na + /K + )ATPase pump activity is no longer needed for progression through the cycle. These data suggests that pump activity is critical in the biosynthetic processes which enables the cell to proceed through the G 1 phase. A scheme is proposed which is currently being tested that (Na + /K + )ATPase pump activity serves as the driving force in the regulation of other membrane transport processes critical for cell proliferation. For example, in post-confluent quiescent C3H-10T1/2 fibroblasts, when [K + ]/sub o/ is lowered just below the K/sub m/ of the pump for K + there is a 10-fold increase in 3 H-uridine uptake into both acid soluble and insoluble cell fractions. By modulation of the pump in this manner, glucose utilization is enhanced whereas inhibition of the pump by ouabain suppresses glucose utilization. In both methods of affecting the pump, 3 H-leucine incorporation is inhibited. Electron acceptors that influence the redox state of the cell have been shown to both stimulate or inhibit cell cycle progression. Under conditions where [K + ]/sub o/ is lowered, the nucleoside uptake responses observed were modified by electron acceptors depending on the ability to oxidize NAD(P)H directly or to interact with a cytochrome-like component, (e.g. phenazine methosulfate) reversed the enhanced uridine uptake and p-phenylene diamine further enhanced the uridine uptake response. These findings suggest that a plasma membrane redox system (presumably cyt-c like) is linked to nucleoside transport which is subject to (Na + /K + )ATPase activity

  16. Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method

    Directory of Open Access Journals (Sweden)

    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.

  17. Functional conservation of coenzyme Q biosynthetic genes among yeasts, plants, and humans.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Hayashi

    Full Text Available Coenzyme Q (CoQ is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3-9 that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

  18. Tuning the pH-shift protein-isolation method for maximum hemoglobin-removal from blood rich fish muscle.

    Science.gov (United States)

    Abdollahi, Mehdi; Marmon, Sofia; Chaijan, Manat; Undeland, Ingrid

    2016-12-01

    A main challenge preventing optimal use of protein isolated from unconventional raw materials (e.g., small pelagic fish and fish by-products) using the pH-shift method is the difficulty to remove enough heme-pigments. Here, the distribution of hemoglobin (Hb) in the different fractions formed during pH-shift processing was studied using Hb-fortified cod mince. Process modifications, additives and prewashing were then investigated to further facilitate Hb-removal. The alkaline pH-shift process version could remove considerably more Hb (77%) compared to the acidic version (37%) when proteins were precipitated at pH 5.5; most Hb was removed during dewatering. Protein precipitation at pH 6.5 improved total Hb removal up to 91% and 74% during alkaline and acid processing, respectively. Adding phytic acid to the first supernatant of the alkaline process version yielded 93% Hb removal. Combining one prewash with phytic acid at pH 5.5 followed by alkaline/acid pH-shift processing increased Hb removal up to 96/92%. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Detection of Ampicillin Resistance Genes (bla in Clinical Isolates of Escherichia coli with Polymerase Chain Reaction Method

    Directory of Open Access Journals (Sweden)

    Tiana Milanda

    2014-09-01

    Full Text Available Escherichia coli is a rod negative Gram which could be pathogenic, if its value increases or located in outer gastrointestinal tract. Pathogenic E. coli will produce enterotoxin which will cause diarrhoea or infection in urine tract. Ampicilin was one of particular antibiotics to overcome infection. Ampicilin nowadays is no longer used as primary medicine, because of its resistance case. The aim of this research is to detect the presence of gene which is responsible to ampicilin resistant E. coli. We used isolated midstream urine from cystitis object in Hasan Sadikin Hospital (RSHS as samples. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were done to invenstigate the antibiotic resistency. Based on the result of antibiotic susceptibility testing to ampicillin, E. coli samples were resistant to ampicilin. Elektroforegram products of colony-PCR and DNA-PCR showed that the resistance case of ampicilin caused by bla gene (199 bp. Selective and rational antibiotic treatment is required to prevent ampicillin resistance in patients with symptoms

  20. Profile of antimicrobial susceptibility isolated microorganisms from hospitalized patients in PICU ward and detection of Methicillin-resistant Staphylococcus aureus and ESBL-producing bacteria by phenotypic methods

    Directory of Open Access Journals (Sweden)

    Shahla Abbas Poor

    2014-10-01

    Full Text Available Background: Hospital-acquired infections are a major challenge to patient. A range of gram-negative organisms are responsible for hospital-acquired infections, the Enterobacteriaceae family being the most commonly identified group overall. Infections by ESBL producers are associated with severe adverse clinical outcomes that have led to increased mortality, prolonged hospitalization, and rising medical costs. The aim of this study was to survey profile of antimicrobial susceptibility isolated microorganisms from hospitalized patients in PICU ward and detection of methicillin-resistant Staphylococcus aureus and ESBL-producing bacteria by phenotypic methods. Material and Methods: In this study participants were patients hospitalized in PICU part of Bahrami Hospital, Tehran, with attention to involved organ. For isolation of bacteria from patient’s samples, culture performed on different selective and differential media. After confirmation of bacteria by biochemical tests, susceptibility testing was performed by disc diffusion method. Phenotypic detection of MRSA strains was performed using cefoxcitin disc. ESBL producing strains were detected by ceftazidime (CAZ and ceftazidime/clavulanic acid (CAZ/CLA discs. Results: Among all isolated organisms from clinical samples, the most common isolated organisms were Escherichia coli (24 cases, Pseudomonas areoginosa (9 cases and Staphylococcus aureus (8 cases, respectively. Among eight MRSA isolated strains from different clinical samples, six strains (75% were MRSA. Among 52 isolated gram negative organisms, 5 strains (9/6% were ESBL. Conclusion: Standard interventions to prevent the transmission of antimicrobial resistance in health care facilities include hand hygiene, using barrier precautions in the care of colonized and infected patients, using dedicated instruments and equipment for these patients. The colonized or infected patients should be isolated in single rooms, multibed rooms or areas

  1. [Isolation and purification of BMScs of GFP transgenic mouse using the method of adhering to cuture plastic in different time].

    Science.gov (United States)

    Li, Fu-Qiang; Zhou, Hong-Ying; Yang, Hui-Lun; Xiang, Tao; Mei, Yan; Hu, Huo-Zhen; Wang, Ting-Hua

    2006-03-01

    To adopt the method of adhering to culture plastic in different time for cultivating and purifying BMSCs of green fluorescent protein (GFP) transgenic mice. Bone marrow cells isolated from GFP transgenic mice are directly planted in culture flask and an exchange of the total volume medium is made at different time. Then the cells adhering to culture plastic are differently counted according to the cell types and are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54 in three days. Moreover, the cells after the exchange of the total volume medium in 4 hours, 8 hours and 24 hours are selected and successively subcultured down to the fifth passage. Then the result of amplification is calculated and the cells are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54. With the extending of the time for the first exchange of medium, the density of cells adhering to culture plastic increased accordingly, but the BMSCs proportion decreased. The cells after first exchange of medium in 4 hours had high BMSCs proportion but low BMSCs density, and the cells in 24 hours had high BMSCs density and low BMSCs proportion. However, the cells in 8-10 hours had high BMSCs density and also high BMSCs proportion. The subcultured BMSCs could stably express GFP. The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to make the first exchange of total volume medium in 8-10 hours. The subcultured cell has the capacity for amplification and will probably be a seed cell for the research of tissue engineering and gene therapy.

  2. Waste Isolation Safety Assessment Program scenario analysis methods for use in assessing the safety of the geologic isolation of nuclear waste

    International Nuclear Information System (INIS)

    Greenborg, J.; Winegardner, W.K.; Pelto, P.J.; Voss, J.W.; Stottlemyre, J.A.; Forbes, I.A.; Fussell, J.B.; Burkholder, H.C.

    1978-11-01

    The relative utility of the various safety analysis methods to scenario analysis for a repository system was evaluated by judging the degree to which certain criteria are satisfied by use of the method. Six safety analysis methods were reviewed in this report for possible use in scenario analysis of nuclear waste repositories: expert opinion, perspectives analysis, fault trees/event trees, Monte Carlo simulation, Markov chains, and classical systems analysis. Four criteria have been selected. The criteria suggest that the methods: (1) be quantitative and scientifically based; (2) model the potential disruptive events and processes, (3) model the system before and after failure (sufficiently detailed to provide for subsequent consequence analysis); and (4) be compatible with the level of available system knowledge and data. Expert opinion, fault trees/event trees, Monte Carlo simulation and classical systems analysis were judged to have the greatest potential appliation to the problem of scenario analysis. The methods were found to be constrained by limited data and by knowledge of the processes governing the system. It was determined that no single method is clearly superior to others when measured against all the criteria. Therefore, to get the best understanding of system behavior, a combination of the methods is recommended. Monte Carlo simulation was judged to be the most suitable matrix in which to incorporate a combination of methods

  3. Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro Biosynthetic Pathway in Streptomyces sp. US24 Strain

    Directory of Open Access Journals (Sweden)

    Samiha Sioud

    2007-01-01

    Full Text Available We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro diketopiperazine (DKP. To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS. The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

  4. Suitability of the molecular subtyping methods intergenic spacer region, direct genome restriction analysis, and pulsed-field gel electrophoresis for clinical and environmental Vibrio parahaemolyticus isolates.

    Science.gov (United States)

    Lüdeke, Catharina H M; Fischer, Markus; LaFon, Patti; Cooper, Kara; Jones, Jessica L

    2014-07-01

    Vibrio parahaemolyticus is the leading cause of infectious illness associated with seafood consumption in the United States. Molecular fingerprinting of strains has become a valuable research tool for understanding this pathogen. However, there are many subtyping methods available and little information on how they compare to one another. For this study, a collection of 67 oyster and 77 clinical V. parahaemolyticus isolates were analyzed by three subtyping methods--intergenic spacer region (ISR-1), direct genome restriction analysis (DGREA), and pulsed-field gel electrophoresis (PFGE)--to determine the utility of these methods for discriminatory subtyping. ISR-1 analysis, run as previously described, provided the lowest discrimination of all the methods (discriminatory index [DI]=0.8665). However, using a broader analytical range than previously reported, ISR-1 clustered isolates based on origin (oyster versus clinical) and had a DI=0.9986. DGREA provided a DI=0.9993-0.9995, but did not consistently cluster the isolates by any identifiable characteristics (origin, serotype, or virulence genotype) and ∼ 15% of isolates were untypeable by this method. PFGE provided a DI=0.9998 when using the combined pattern analysis of both restriction enzymes, SfiI and NotI. This analysis was more discriminatory than using either enzyme pattern alone and primarily grouped isolates by serotype, regardless of strain origin (clinical or oyster) or presence of currently accepted virulence markers. These results indicate that PFGE and ISR-1 are more reliable methods for subtyping V. parahemolyticus, rather than DGREA. Additionally, ISR-1 may provide an indication of pathogenic potential; however, more detailed studies are needed. These data highlight the diversity within V. parahaemolyticus and the need for appropriate selection of subtyping methods depending on the study objectives.

  5. The Biosynthetic Origin of Irregular Monoterpenes in Lavandula

    Science.gov (United States)

    Demissie, Zerihun A.; Erland, Lauren A. E.; Rheault, Mark R.; Mahmoud, Soheil S.

    2013-01-01

    Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s−1, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering. PMID:23306202

  6. Identification of Echinococcus Granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan – Iran

    Directory of Open Access Journals (Sweden)

    Haleh Hanifian

    2013-09-01

    Full Text Available Background: The aim of this study was DNA extraction from protosco­lecses of Echinococcus granulosus and identification of these strains in West-Azerbai­jan Province, north western Iran.Methods: Thirty one livestock isolates from sheep and cattle were collected from abattoirs of the province. To investigate the genetic variation of the isolates, after DNA extraction by Glass beads-phenol chloroform method; PCR-RLFP analysis of rDNA-ITS1 was performed using three different restric­tion enzymes of Taq 1, Rsa 1 and Alu 1.Result: Amplified PCR products for all isolates were 1000bp band which is expected band in sheep strains (G1-G3 complex. The results of RFLP analy­sis also were the same for all isolates. PCR-RFLP patterns restriction en­zymes were identical as follows, Rsa1 bands under UV showed two bands approximately 655bp and 345bp. Alu1 bands were as follows: two approx­imately 800bp and 200bp and Taq1 did not cut any region and bands were approximately 1000 bp in all samples.Conclusions: Based on PCR-RFLP patterns of ITS1 fragment produced with endonucleases enzyme digestion in animal isolates, it can be concluded that a single strain of E. granulosus (sheep strain or G1-G3 complex is domi­nant genotype in this province

  7. Isolation of Inositol Hexaphosphate (IHP)-Degrading Bacteria from Arbuscular Mycorrhizal Fungal Hyphal Compartments Using a Modified Baiting Method Involving Alginate Beads Containing IHP

    Science.gov (United States)

    Hara, Shintaro; Saito, Masanori

    2016-01-01

    Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF. PMID:27383681

  8. Shake and stew: a non-destructive PCR-ready DNA isolation method from a single preserved fish larva.

    Science.gov (United States)

    Alvarado Bremer, J R; Smith, B L; Moulton, D L; Lu, C-P; Cornic, M

    2014-01-01

    A rapid non-destructive alternative to isolate DNA from an individual fish larva is presented, based on the suspension of epithelial cells through vortex forces, and the release of DNA in a heated alkaline solution. DNA from >6056 fish larvae isolated using this protocol has yielded a high PCR amplification success rate (>93%), suggesting its applicability to other taxonomic groups or sources when tissue amount is the limiting factor. © 2014 The Fisheries Society of the British Isles.

  9. Evaluation of a Microculture Method for Isolation of Leishmania Parasites from Cutaneous Lesions of Patients in Peru▿

    Science.gov (United States)

    Boggild, Andrea K.; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E.

    2007-01-01

    Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-μl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture. PMID:17881557

  10. Evaluation of a microculture method for isolation of Leishmania parasites from cutaneous lesions of patients in Peru.

    Science.gov (United States)

    Boggild, Andrea K; Miranda-Verastegui, Cesar; Espinosa, Diego; Arevalo, Jorge; Adaui, Vanessa; Tulliano, Gianfranco; Llanos-Cuentas, Alejandro; Low, Donald E

    2007-11-01

    Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-microl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture.

  11. Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

    Science.gov (United States)

    Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

    2017-11-30

    The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

  12. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae.

    Science.gov (United States)

    Lin-Wang, Kui; Bolitho, Karen; Grafton, Karryn; Kortstee, Anne; Karunairetnam, Sakuntala; McGhie, Tony K; Espley, Richard V; Hellens, Roger P; Allan, Andrew C

    2010-03-21

    The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

  13. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

    Directory of Open Access Journals (Sweden)

    McGhie Tony K

    2010-03-01

    Full Text Available Abstract Background The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry. Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

  14. The preparation of nucleotides uniformly labelled with carbon-14 by biosinthetic methods. Isolation of adenilic, uridilic, cytidilic and guanlic acids, from the alkaline hydrolisate of escherichia coli RNA

    International Nuclear Information System (INIS)

    Garcia Pineda, D.; Pacheco Lopez, J.

    1978-01-01

    A method is described for the preparation and analysis of adenilic, uridilic, cytidylic and guanilic acids, labelled with carbon 14. Escherichia coli cells have been labelled by growing them in media containing glucose-carbon 14 as their only source of carbon. RNA is isolated from the cells, and after hydrolisis of the molecule the resulting nucleotides are separated by gel filtration and exchange chromatography. Chemical and radiochemical purity of the isolated nucleotides is determined and also its specific radioactivity. The distribution of radioactivity incorporated in the cell among different groups of molecular species is analyse. (author)

  15. An accelerated method for the detection of Extended-Spectrum B- Lactamases in urinary isolates of Escherichia Coli and Klebsiella pneumoniae

    International Nuclear Information System (INIS)

    Kader, Abdulrahman A.; Kumar, A.; Krishna, A.; Zaman, M.N.

    2006-01-01

    We prospectively studied an accelerated phenotypic method by incorporating the double disk synergy test in the standard Kirby-Bauer disk diffusion susceptibility testing, to evaluate a protocol for the rapid detection of extended of extended-spectrum B-lactamases (ESBL) in urinary isolates of Escherichia coli (E. coli) and Klebsiella, pneumoniae (K. pneumoniae). All ESBL-positive isolates were confirmed by the standard Clinical Laboratory Standards Institute (CLSI) confirmatory disk diffusion method. Between November 2004 and December 2005, a total of 6988 urine specimens were analyzed of which 776 (11%) showed significant growth. They included E. coli in 577 cases (74%) and K. pneumoniae in 199 (25.6%). Of these, 63 E. coli (8%) and 15 K. pneumoniae (7.5%) were positive for ESBL by the accelerated and CLSI methods. Compared to the standard CLSI method, the accelerated method reduced the ESBL detection time from two days to one day. We conclude that the accelerated ESBL detection technique used by us in this study is a reliable and rapid method for detecting ESBL in urinary isolates of E. coli and K. pneumoniae. (author)

  16. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201.

    Science.gov (United States)

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George

    2011-01-01

    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  17. Evaluation of Etest and macrodilution broth method for antifungal susceptibility testing of Candida sp strains isolated from oral cavities of AIDS patients

    Directory of Open Access Journals (Sweden)

    SILVA Maria do Rosário R.

    2002-01-01

    Full Text Available A comparison of the Etest and the reference broth macrodilution susceptibility test for fluconazole, ketoconazole, itraconazole and amphotericin B was performed with 59 of Candida species isolated from the oral cavities of AIDS patients. The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines. Our data showed that there was a good correlation between the MICs obtained by the Etest and broth dilution methods. When only the MIC results at ± 2 dilutions for both methods were considered, the agreement rates were 90.4% for itraconazole, ketoconazole and amphotericin B and 84.6% for fluconazole of the C. albicans tested. In contrast, to the reference method, the Etest method classified as susceptible three fluconazole-resistant isolates and one itraconazole-resistant isolate, representing four very major errors. These results indicate that Etest could be considered useful for antifungal sensitivity evaluation of yeasts in clinical laboratories.

  18. A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant, Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang

    2016-07-01

    Full Text Available Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from E. sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase and EsFLS (flavonol synthase, but not the promoters of EsDFRs (dihydroflavonol 4-reductase and EsANS (anthocyanidin synthase in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase, NtCHI (chalcone isomerase, NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived bioactive components in E

  19. Social isolation and loneliness in later life: A parallel convergent mixed-methods case study of older adults and their residential contexts in the Minneapolis metropolitan area, USA.

    Science.gov (United States)

    Finlay, Jessica M; Kobayashi, Lindsay C

    2018-07-01

    Social isolation and loneliness are increasingly prevalent among older adults in the United States, with implications for morbidity and mortality risk. Little research to date has examined the complex person-place transactions that contribute to social well-being in later life. This study aimed to characterize personal and neighborhood contextual influences on social isolation and loneliness among older adults. Interviews were conducted with independent-dwelling men and women (n = 124; mean age 71 years) in the Minneapolis metropolitan area (USA) from June to October, 2015. A convergent mixed-methods design was applied, whereby quantitative and qualitative approaches were used in parallel to gain simultaneous insights into statistical associations and in-depth individual perspectives. Logistic regression models predicted self-reported social isolation and loneliness, adjusted for age, gender, past occupation, race/ethnicity, living alone, street type, residential location, and residential density. Qualitative thematic analyses of interview transcripts probed individual experiences with social isolation and loneliness. The quantitative results suggested that African American adults, those with a higher socioeconomic status, those who did not live alone, and those who lived closer to the city center were less likely to report feeling socially isolated or lonely. The qualitative results identified and explained variation in outcomes within each of these factors. They provided insight on those who lived alone but did not report feeling lonely, finding that solitude was sought after and enjoyed by a portion of participants. Poor physical and mental health often resulted in reporting social isolation, particularly when coupled with poor weather or low-density neighborhoods. At the same time, poor health sometimes provided opportunities for valued social engagement with caregivers, family, and friends. The combination of group-level risk factors and in-depth personal

  20. Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for Campylobacters

    DEFF Research Database (Denmark)

    Engberg, J.; On, Stephen L.W.; Harrington, C.S.

    2000-01-01

    for isolation of Campylobacter spp. Two charcoal-based selective media, modified charcoal cefoperazone deoxycholate agar (mCCDA) and cefoperazone-amphotericin-teicoplanin (CAT) agar, were compared with Skirrow's blood-based medium and with a filter method (filter) applied to a yeast-enriched blood agar. A total...... of 1,376 specimens were tested on all four media, and the percentages of thermophilic Campylobacter-positive specimens isolated on Skirrow's medium, filters, CAT agar, and mCCDA were 82, 83, 85, and 95%, respectively. When additional samples were professed with the three selective media, m...... butzleri, Arcobacter cryaerophilus, Helicobacter cinaedi, and Sutterella wadsworthensis. Most of these strains were isolated after 5 to 6 days of incubation by use of the filter technique. This paper pro,ides evidence for the existence of S. wadsworthensis in human feces from clinical cases...

  1. Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

    DEFF Research Database (Denmark)

    Knüsel, R.; Bergmann, S. M.; Einer-Jensen, Katja

    2007-01-01

    in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus......This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools...... (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and...

  2. Isolation of bacterial cellulose nanocrystalline from pineapple peel waste: Optimization of acid concentration in the hydrolysis method

    Science.gov (United States)

    Anwar, Budiman; Rosyid, Nurul Huda; Effendi, Devi Bentia; Nandiyanto, Asep Bayu Dani; Mudzakir, Ahmad; Hidayat, Topik

    2016-02-01

    Isolation of needle-shaped bacterial cellulose nanocrystalline with a diameter of 16-64 nm, a fiber length of 258-806 nm, and a degree of crystallinity of 64% from pineapple peel waste using an acid hydrolysis process was investigated. Experimental showed that selective concentration of acid played important roles in isolating the bacterial cellulose nanocrystalline from the cellulose source. To achieve the successful isolation of bacterial cellulose nanocrystalline, various acid concentrations were tested. To confirm the effect of acid concentration on the successful isolation process, the reaction conditions were fixed at a temperature of 50°C, a hydrolysis time of 30 minutes, and a bacterial cellulose-to-acid ratio of 1:50. Pineapple peel waste was used as a model for a cellulose source because to the best of our knowledge, there is no report on the use of this raw material for producing bacterial cellulose nanocrystalline. In fact, this material can be used as an alternative for ecofriendly and cost-free cellulose sources. Therefore, understanding in how to isolate bacterial cellulose nanocrystalline from pineapple peel waste has the potential for large-scale production of inexpensive cellulose nanocrystalline.

  3. Diurnal and circadian expression profiles of glycerolipid biosynthetic genes in Arabidopsis.

    Science.gov (United States)

    Nakamura, Yuki; Andrés, Fernando; Kanehara, Kazue; Liu, Yu-chi; Coupland, George; Dörmann, Peter

    2014-01-01

    Glycerolipid composition in plant membranes oscillates in response to diurnal change. However, its functional significance remained unclear. A recent discovery that Arabidopsis florigen FT binds diurnally oscillating phosphatidylcholine molecules to promote flowering suggests that diurnal oscillation of glycerolipid composition is an important input in flowering time control. Taking advantage of public microarray data, we globally analyzed the expression pattern of glycerolipid biosynthetic genes in Arabidopsis under long-day, short-day, and continuous light conditions. The results revealed that 12 genes associated with glycerolipid metabolism showed significant oscillatory profiles. Interestingly, expression of most of these genes followed circadian profiles, suggesting that glycerolipid biosynthesis is partially under clock regulation. The oscillating expression profile of one representative gene, PECT1, was analyzed in detail. Expression of PECT1 showed a circadian pattern highly correlated with that of the clock-regulated gene GIGANTEA. Thus, our study suggests that a considerable number of glycerolipid biosynthetic genes are under circadian control.

  4. In silico tools for the analysis of antibiotic biosynthetic pathways

    DEFF Research Database (Denmark)

    Weber, Tilmann

    2014-01-01

    Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel...... screening methods based on the genome sequences of potential producing organisms. The basic principle of such genome mining approaches is to identify genes, which are involved in the biosynthesis of such molecules, and to predict the products of the identified pathways. Thus, bioinformatics methods...... and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data....

  5. Identification of strains with phenotypes similar to those of Staphylococcus aureus isolated from table chicken eggs using MALDI-TOF MS and genotyping methods

    Directory of Open Access Journals (Sweden)

    Marek Agnieszka

    2015-06-01

    Full Text Available The aim of the study was to identify the affinity of 10 Staphylococcus strains isolated from table chicken eggs to specific species. Preliminary analysis performed by API ID32 Staph test identified these strains as S. aureus, but they exhibited a negative reaction in the tube coagulase test. Thus, the analysed strains were initially characterised as Staphylococcus aureus-like (SAL. Further characterisation was performed by genotypic methods, using restriction fragment length polymorphism (RFLP of the coagulase gene (coa and sequencing of the gene rpoB. An attempt was also made to identify the isolated Staphylococcus strains by MALDI-TOF mass spectrometry. The results indicated that none of the strains tested belonged to the species S. aureus. The rpoB sequences of five isolates showed the highest sequence similarity to S. haemolyticus, three isolates to S. chromogenes, and one isolate to S. epidermidis. One strain (SAL4 remained unidentified in this analysis. The results obtained using mass spectrometry were comparable to those based on gene sequence analysis. Strain SAL4, which could not be identified by sequencing, was identified by MALDI-TOF as Staphylococcus chromogenes.

  6. Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c

    Directory of Open Access Journals (Sweden)

    José J. Fernández

    2012-10-01

    Full Text Available The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP. In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of 13C enriched samples obtained by addition of labelled sodium [l-13C], [2-13C] acetate to artificial cultures of this dinoflagellate.

  7. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera

    DEFF Research Database (Denmark)

    Ono, Hajime; Rewitz, Kim; Shinoda, Tetsu

    2006-01-01

    is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both...... Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development....

  8. Linking metabolic QTLs with network and cis-eQTLs controlling biosynthetic pathways.

    Directory of Open Access Journals (Sweden)

    Adam M Wentzell

    2007-09-01

    Full Text Available Phenotypic variation between individuals of a species is often under quantitative genetic control. Genomic analysis of gene expression polymorphisms between individuals is rapidly gaining popularity as a way to query the underlying mechanistic causes of variation between individuals. However, there is little direct evidence of a linkage between global gene expression polymorphisms and phenotypic consequences. In this report, we have mapped quantitative trait loci (QTLs-controlling glucosinolate content in a population of 403 Arabidopsis Bay x Sha recombinant inbred lines, 211 of which were previously used to identify expression QTLs controlling the transcript levels of biosynthetic genes. In a comparative study, we have directly tested two plant biosynthetic pathways for association between polymorphisms controlling biosynthetic gene transcripts and the resulting metabolites within the Arabidopsis Bay x Sha recombinant inbred line population. In this analysis, all loci controlling expression variation also affected the accumulation of the resulting metabolites. In addition, epistasis was detected more frequently for metabolic traits compared to transcript traits, even when both traits showed similar distributions. An analysis of candidate genes for QTL-controlling networks of transcripts and metabolites suggested that the controlling factors are a mix of enzymes and regulatory factors. This analysis showed that regulatory connections can feedback from metabolism to transcripts. Surprisingly, the most likely major regulator of both transcript level for nearly the entire pathway and aliphatic glucosinolate accumulation is variation in the last enzyme in the biosynthetic pathway, AOP2. This suggests that natural variation in transcripts may significantly impact phenotypic variation, but that natural variation in metabolites or their enzymatic loci can feed back to affect the transcripts.

  9. Pulmonary vein isolation using a second-generation cryoballoon catheter: a randomized comparison of ablation duration and method of deflation.

    Science.gov (United States)

    Andrade, Jason G; Dubuc, Marc; Guerra, Peter G; Landry, Evelyn; Coulombe, Nicolas; Leduc, Hugues; Rivard, Léna; Macle, Laurent; Thibault, Bernard; Talajic, Mario; Roy, Denis; Khairy, Paul

    2013-06-01

    Optimal cryoballoon ablation parameters for pulmonary vein (PV) isolation remain to be defined. We conducted a randomized preclinical trial to compare 2- versus 4-minute ablation lesions and assess the safety of active (forced) cryoballoon deflation. Thirty-two dogs underwent PV isolation with a second-generation 23 mm cryoballoon catheter. The left superior (LSPV) and inferior (LIPV) PVs were randomized in a factorial design to (1) a single 2- versus 4-minute cryoapplication, and (2) passive versus active cryoballoon deflation. Animals were survived for 30 days, after which histopathologic analysis was performed. Acute PV isolation was attained in 89.8% of PVs after a single application (93.8% LSPV, 85.2% LIPV; P = 0.2823). Mean time to PV isolation was 29.5 ± 18.5 seconds. Although 4-minute lesions were associated with a thicker neointima than 2-minute lesions (223.8 μm versus 135.6 μm; P = 0.007), no differences were observed in procedural characteristics (freezing temperature, rewarming time), rates of acute PV isolation, or the achievement of complete circumferentially transmural lesions at 30 days (78.7% overall; 86.2% for 2 minutes vs 70.0% for 4 minutes; P = 0.285). Active deflation was associated with faster balloon rewarming but not with significant differences in mean or maximum neointimal thickness. A single application with the second-generation cryoballoon catheter results in a high rate of PV isolation. The degree of vascular injury was not increased by active balloon deflation and no differences in acute efficacy or mature transmural circumferential lesions were observed with 2- versus 4-minute applications. © 2013 Wiley Periodicals, Inc.

  10. A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

    Directory of Open Access Journals (Sweden)

    Borui Pi

    Full Text Available Secondary metabolites (SMs produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

  11. A simple biosynthetic pathway for large product generation from small substrate amounts

    Science.gov (United States)

    Djordjevic, Marko; Djordjevic, Magdalena

    2012-10-01

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways.

  12. Enhancement of Nucleoside Production in Hirsutella sinensis Based on Biosynthetic Pathway Analysis

    Science.gov (United States)

    Liu, Zhi-Qiang; Zhang, Bo; Lin, Shan; Baker, Peter James; Chen, Mao-Sheng; Xue, Ya-Ping; Wu, Hui; Xu, Feng; Yuan, Shui-Jin; Teng, Yi; Wu, Ling-Fang

    2017-01-01

    To enhance nucleoside production in Hirsutella sinensis, the biosynthetic pathways of purine and pyrimidine nucleosides were constructed and verified. The differential expression analysis showed that purine nucleoside phosphorylase, inosine monophosphate dehydrogenase, and guanosine monophosphate synthase genes involved in purine nucleotide biosynthesis were significantly upregulated 16.56-fold, 8-fold, and 5.43-fold, respectively. Moreover, dihydroorotate dehydrogenase, uridine nucleosidase, uridine/cytidine monophosphate kinase, and inosine triphosphate pyrophosphatase genes participating in pyrimidine nucleoside biosynthesis were upregulated 4.53-fold, 10.63-fold, 4.26-fold, and 5.98-fold, respectively. To enhance the nucleoside production, precursors for synthesis of nucleosides were added based on the analysis of biosynthetic pathways. Uridine and cytidine contents, respectively, reached 5.04 mg/g and 3.54 mg/g when adding 2 mg/mL of ribose, resulting in an increase of 28.6% and 296% compared with the control, respectively. Meanwhile, uridine and cytidine contents, respectively, reached 10.83 mg/g 2.12 mg/g when adding 0.3 mg/mL of uracil, leading to an increase of 176.3% and 137.1%, respectively. This report indicated that fermentation regulation was an effective way to enhance the nucleoside production in H. sinensis based on biosynthetic pathway analysis. PMID:29333435

  13. A simple biosynthetic pathway for large product generation from small substrate amounts

    Energy Technology Data Exchange (ETDEWEB)

    Djordjevic, Marko [Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade (Serbia); Djordjevic, Magdalena [Institute of Physics Belgrade, University of Belgrade (Serbia)

    2012-10-01

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways. (paper)

  14. Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis.

    Science.gov (United States)

    Lin, Shan; Liu, Zhi-Qiang; Baker, Peter James; Yi, Ming; Wu, Hui; Xu, Feng; Teng, Yi; Zheng, Yu-Guo

    2016-11-01

    The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli.

    Science.gov (United States)

    Stahlhut, Steen G; Siedler, Solvej; Malla, Sailesh; Harrison, Scott J; Maury, Jérôme; Neves, Ana Rute; Forster, Jochen

    2015-09-01

    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  16. Fabrication of biosynthetic vascular prostheses by 193-nm excimer laser radiation

    Science.gov (United States)

    Husinsky, Wolfgang; Csek, Ch.; Bartel, A.; Grabenwoeger, M.; Fitzal, F.; Wolner, Ernst

    1998-05-01

    This study was undertaken to investigate the feasibility of transmural capillary ingrowth into the inner surface of biosynthetic vascular prostheses (OmniflowTM) through perforations created by an excimer-laser, thus inducing an endothelial cell coverage. The biosynthetic vascular prostheses (10 cm length, 6 mm (phi) ) were perforated with an excimer laser ((phi) of the holes 50 - 100 micrometer, distance 4 mm) and implanted into the carotid arteries of 8 sheep. The laser tissue interaction process of 193 nm radiation ensures minimal thermal damage to the prostheses. They were compared to untreated OmniflowTM prostheses implanted at the contralateral side. Three months after implantation the prostheses were explanted and evaluated by gross morphology, histological examination and scanning electron microscopy. Scanning electron microscopy showed endothelial cells in the midgraft portion of all perforated prostheses, whereas collagen fibers, fibrin meshwork and activated platelets formed the inner layer in 6 out of 8 untreated OmniflowTM prostheses. It can be concluded, that spontaneous endothelialization of biosynthetic vascular prostheses can be achieved by transmural capillary ingrowth through perforations in the wall of the prostheses in an experimental sheep model.

  17. A simple biosynthetic pathway for large product generation from small substrate amounts

    International Nuclear Information System (INIS)

    Djordjevic, Marko; Djordjevic, Magdalena

    2012-01-01

    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways. (paper)

  18. A Genomics Based Discovery of Secondary Metabolite Biosynthetic Gene Clusters in Aspergillus ustus

    Science.gov (United States)

    Pi, Borui; Yu, Dongliang; Dai, Fangwei; Song, Xiaoming; Zhu, Congyi; Li, Hongye; Yu, Yunsong

    2015-01-01

    Secondary metabolites (SMs) produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic. PMID:25706180

  19. Draft Genome Sequence of Marine Sponge Symbiont Pseudoalteromonas luteoviolacea IPB1, Isolated from Hilo, Hawaii.

    Science.gov (United States)

    Sakai-Kawada, Francis E; Yakym, Christopher J; Helmkampf, Martin; Hagiwara, Kehau; Ip, Courtney G; Antonio, Brandi J; Armstrong, Ellie; Ulloa, Wesley J; Awaya, Jonathan D

    2016-09-22

    We report here the 6.0-Mb draft genome assembly of Pseudoalteromonas luteoviolacea strain IPB1 that was isolated from the Hawaiian marine sponge Iotrochota protea Genome mining complemented with bioassay studies will elucidate secondary metabolite biosynthetic pathways and will help explain the ecological interaction between host sponge and microorganism. Copyright © 2016 Sakai-Kawada et al.

  20. Identification and analysis of the paulomycin biosynthetic gene cluster and titer improvement of the paulomycins in Streptomyces paulus NRRL 8115.

    Directory of Open Access Journals (Sweden)

    Jine Li

    Full Text Available The paulomycins are a group of glycosylated compounds featuring a unique paulic acid moiety. To locate their biosynthetic gene clusters, the genomes of two paulomycin producers, Streptomyces paulus NRRL 8115 and Streptomyces sp. YN86, were sequenced. The paulomycin biosynthetic gene clusters were defined by comparative analyses of the two genomes together with the genome of the third paulomycin producer Streptomyces albus J1074. Subsequently, the identity of the paulomycin biosynthetic gene cluster was confirmed by inactivation of two genes involved in biosynthesis of the paulomycose branched chain (pau11 and the ring A moiety (pau18 in Streptomyces paulus NRRL 8115. After determining the gene cluster boundaries, a convergent biosynthetic model was proposed for paulomycin based on the deduced functions of the pau genes. Finally, a paulomycin high-producing strain was constructed by expressing an activator-encoding gene (pau13 in S. paulus, setting the stage for future investigations.

  1. Recent development of antiSMASH and other computational approaches to mine secondary metabolite biosynthetic gene clusters

    DEFF Research Database (Denmark)

    Blin, Kai; Kim, Hyun Uk; Medema, Marnix H.

    2017-01-01

    Many drugs are derived from small molecules produced by microorganisms and plants, so-called natural products. Natural products have diverse chemical structures, but the biosynthetic pathways producing those compounds are often organized as biosynthetic gene clusters (BGCs) and follow a highly...... conserved biosynthetic logic. This allows for the identification of core biosynthetic enzymes using genome mining strategies that are based on the sequence similarity of the involved enzymes/genes. However, mining for a variety of BGCs quickly approaches a complexity level where manual analyses...... are no longer possible and require the use of automated genome mining pipelines, such as the antiSMASH software. In this review, we discuss the principles underlying the predictions of antiSMASH and other tools and provide practical advice for their application. Furthermore, we discuss important caveats...

  2. Effect of overall feedback inhibition in unbranched biosynthetic pathways.

    Science.gov (United States)

    Alves, R; Savageau, M A

    2000-11-01

    We have determined the effects of control by overall feedback inhibition on the systemic behavior of unbranched metabolic pathways with an arbitrary pattern of other feedback inhibitions by using a recently developed numerical generalization of Mathematically Controlled Comparisons, a method for comparing the function of alternative molecular designs. This method allows the rigorous determination of the changes in systemic properties that can be exclusively attributed to overall feedback inhibition. Analytical results show that the unbranched pathway can achieve the same steady-state flux, concentrations, and logarithmic gains with respect to changes in substrate, with or without overall feedback inhibition. The analytical approach also shows that control by overall feedback inhibition amplifies the regulation of flux by the demand for end product while attenuating the sensitivity of the concentrations to the same demand. This approach does not provide a clear answer regarding the effect of overall feedback inhibition on the robustness, stability, and transient time of the pathway. However, the generalized numerical method we have used does clarify the answers to these questions. On average, an unbranched pathway with control by overall feedback inhibition is less sensitive to perturbations in the values of the parameters that define the system. The difference in robustness can range from a few percent to fifty percent or more, depending on the length of the pathway and on the metabolite one considers. On average, overall feedback inhibition decreases the stability margins by a minimal amount (typically less than 5%). Finally, and again on average, stable systems with overall feedback inhibition respond faster to fluctuations in the metabolite concentrations. Taken together, these results show that control by overall feedback inhibition confers several functional advantages upon unbranched pathways. These advantages provide a rationale for the prevalence of this

  3. A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours.

    Science.gov (United States)

    Reddy, Divya; Khade, Bharat; Pandya, Riddhi; Gupta, Sanjay

    2017-01-01

    Dysregulation in post-translational modifications of histones and their modifiers are now well-recognized as a hallmark of cancer and can be used as biomarkers and potential therapeutic targets for disease progression and prognosis. In most solid tumours, a biopsy is challenging, costly, painful or potentially risky for the patient. Therefore, non-invasive methods like 'liquid biopsy' for analysis of histone modifications and their modifiers if possible will be helpful in the better clinical management of cancer patients. Here, we have developed a cost-effective and time-efficient protocol for isolation of circulating histones from serum of solid tumor, HCC, called Dual Acid Extraction (DAE) protocol and have confirmed by mass spectrometry. Also, we measured the activity of HDACs and HATs in serum samples. The serum purified histones were profiled for changes in histone PTMs and have shown a comparable pattern of modifications like acetylation (H4K16Ac), methylation (H4K20Me3, H3K27Me3, H3K9Me3) and phosphorylation (γ-H2AX and H3S10P) to paired cancer tissues. Profiling for the histone PTM changes in various other organs of normal and tumor bearing animal suggests that the changes in the histone PTMs observed in the tumor serum is indeed due to changes in the tumor tissue only. Further, we demonstrate that the observed hypo-acetylation of histone H4 in tissue and serum samples of tumor bearing animals corroborated with the elevated HDAC activity in both samples compared to normal. Interestingly, human normal and tumor serum samples also showed elevated HDAC activity with no significant changes in HAT activity. Our study provides the first evidence in the context of histone PTMs and modifiers that liquid biopsy is a valuable predictive tool for monitoring disease progression. Importantly, with the advent of drugs that target specific enzymes involved in the epigenetic regulation of gene expression, liquid biopsy-based 'real time' monitoring will be useful for

  4. Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR.

    Science.gov (United States)

    Jribi, Hela; Sellami, Hanen; Mariam, Siala; Smaoui, Salma; Ghorbel, Asma; Hachicha, Salma; Benejat, Lucie; Messadi-Akrout, Feriel; Mégraud, Francis; Gdoura, Radhouane

    2017-10-01

    Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli . Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli . All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked

  5. Bacterial natural product biosynthetic domain composition in soil correlates with changes in latitude on a continent-wide scale.

    Science.gov (United States)

    Lemetre, Christophe; Maniko, Jeffrey; Charlop-Powers, Zachary; Sparrow, Ben; Lowe, Andrew J; Brady, Sean F

    2017-10-31

    Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of small-molecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts. Published under the PNAS license.

  6. Use of Limiting Dilution Method for Isolation of Nucleus Pulposus Mesenchymal Stem/Progenitor Cells and Effects of Plating Density on Biological Characteristics and Plasticity

    Directory of Open Access Journals (Sweden)

    Linghan Lin

    2017-01-01

    Full Text Available Objectives. To evaluate the effects of the limiting dilution method and plating density in rat nucleus pulposus mesenchymal stem/progenitor cells (NPMSCs. Materials and Methods. Nucleus pulposus tissues were isolated from 12-week-old male Sprague-Dawley rats and NPMSCs were isolated using limiting dilution method. Cells were then classified into 3 groups according to plating density. Cell morphologies were observed, and colony-forming units, migration abilities, proliferative capacities, cell cycle percentages, multilineage differentiation capacities, stem cell biomarker expression levels, and immunophenotyping were also examined in each group. Results. Low density group (LD had higher morphological homogeneity, stronger colony-forming ability, higher cell proliferation capacity, and enhanced cell migration ability relative to the other two groups (p<0.05. Moreover, LD had more cells entering S phase, with fewer cells arrested in G0/G1 phase (p<0.05. While all three density groups showed a multilineage differentiation potential, LD showed a higher degree of observed and semiquantified lineage specific staining (p<0.05. Furthermore, LD displayed higher expression levels of stem cell biomarkers (Nanog, Oct4, and Sox2 and showed higher percentages of CD29+, CD44+, and CD90+ cells (p<0.05 following flow cytometry analysis. Conclusions. Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.

  7. HPTLC and reverse phase HPLC methods for the simultaneous quantification and in vitro screening of antioxidant potential of isolated sesquiterpenoids from the rhizomes of Cyperus rotundus.

    Science.gov (United States)

    Priya Rani, M; Padmakumari, K P

    2012-09-01

    Three sesquiterpenoids solavetivone, aristolone and nootkatone were isolated from the acetone extract of Cyperus rotundus by silica gel column chromatography and identified by spectral studies. Solavetivone has been isolated for the first time from the species. Simple, sensitive and selective HPTLC and HPLC methods with ultraviolet detection (245 nm) were developed and validated for the simultaneous quantification. HPTLC method was validated in terms of their linearity, LOD, LOQ, precision, accuracy and compared with RP-HPLC-UV method. Among the three sesquiterpenoids isolated, nootkatone possessed the highest radical scavenging potential (IC(50) 4.81 μg/ml) followed by aristolone (IC(50) 5.28 μg/ml) and solavetivone (IC(50) 6.82 μg/ml) by DPPH radical scavenging assay. Total antioxidant activity against phosphomolybdenum reagent was also studied. The methods described in this paper were able to identify and quantify sesquiterpenoids from the complex mixtures of phytochemicals and could be extended to the marker based standardization of polyherbal formulations containing C. rotundus. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Design and methods of a social network isolation study for reducing respiratory infection transmission: The eX-FLU cluster randomized trial.

    Science.gov (United States)

    Aiello, Allison E; Simanek, Amanda M; Eisenberg, Marisa C; Walsh, Alison R; Davis, Brian; Volz, Erik; Cheng, Caroline; Rainey, Jeanette J; Uzicanin, Amra; Gao, Hongjiang; Osgood, Nathaniel; Knowles, Dylan; Stanley, Kevin; Tarter, Kara; Monto, Arnold S

    2016-06-01

    Social networks are increasingly recognized as important points of intervention, yet relatively few intervention studies of respiratory infection transmission have utilized a network design. Here we describe the design, methods, and social network structure of a randomized intervention for isolating respiratory infection cases in a university setting over a 10-week period. 590 students in six residence halls enrolled in the eX-FLU study during a chain-referral recruitment process from September 2012-January 2013. Of these, 262 joined as "seed" participants, who nominated their social contacts to join the study, of which 328 "nominees" enrolled. Participants were cluster-randomized by 117 residence halls. Participants were asked to respond to weekly surveys on health behaviors, social interactions, and influenza-like illness (ILI) symptoms. Participants were randomized to either a 3-Day dorm room isolation intervention or a control group (no isolation) upon illness onset. ILI cases reported on their isolation behavior during illness and provided throat and nasal swab specimens at onset, day-three, and day-six of illness. A subsample of individuals (N=103) participated in a sub-study using a novel smartphone application, iEpi, which collected sensor and contextually-dependent survey data on social interactions. Within the social network, participants were significantly positively assortative by intervention group, enrollment type, residence hall, iEpi participation, age, gender, race, and alcohol use (all Pisolation from social networks in a university setting. These data provide an unparalleled opportunity to address questions about isolation and infection transmission, as well as insights into social networks and behaviors among college-aged students. Several important lessons were learned over the course of this project, including feasible isolation durations, the need for extensive organizational efforts, as well as the need for specialized programmers and server

  9. Characterization of multi-drug resistant ESBL producing nonfermenter bacteria isolated from patients blood samples using phenotypic methods in Shiraz (Iran

    Directory of Open Access Journals (Sweden)

    Maneli Amin Shahidi

    2015-10-01

    Full Text Available Background and Aim: The emergence of  nonfermenter bacteria that are resistant to multidrug resistant ESBL  are  nowadays a principal problem  for hospitalized patients. The present study aimed at surveying the emergence of nonfermenter bacteria resistant to multi-drug ESBL producing isolated from patients blood samples using BACTEC 9240 automatic system in Shiraz. Materials and Methods: In this cross-sectional study, 4825 blood specimens were collected from hospitalized patients in Shiraz (Iran, and positive samples were detected by means of  BACTEC 9240 automatic system. The isolates  containing nonfermenter bacteria were identified based on biochemical tests embedded in the API-20E system. Antibiotic sensitivity  test was performed  and identification of  ESBL producing strains were done  using phenotypic detection of extended spectrum beta-lactamase producing isolates(DDST according to CLSI(2013 guidelines.   Results: Out of 4825 blood samples, 1145 (24% specimen were gram-positive using BACTEC system. Among all isolated microorganisms, 206 isolates were non-fermenting gram- negative bacteria. The most common non-fermenter isolates were Pseudomonas spp. (48%, Acinetobacter spp. (41.7% ,and Stenotrophomonas spp. (8.2%. Seventy of them (81.4% were  Acinetobacter spp. which were ESBL positive. Among &beta-lactam antibiotics, Pseudomonas spp. showed  the best sensitivity to piperacillin-tazobactam (46.5%.  Conclusion: It was found that  &beta-lactam antibiotics are not effective against more than 40% of Pseudomonas spp. infections and 78% Acinetobacter infections. Emergence of multi-drug resistant strains that are resistant to most antibiotic classes is a major public health problem in Iran. To resolve this problem using of practical guidelines is critical.

  10. Isolation of Notoamide S and Enantiomeric 6-epi-Stephacidin A from the Fungus Aspergillus amoenus: Biogenetic Implications

    DEFF Research Database (Denmark)

    Kato, Hikaru; Nakahara, Takashi; Sugimoto, Kayo

    2015-01-01

    Notoamide S has been hypothesized to be a key biosynthetic intermediate for characteristic metabolites stephacidin A, notoamide B, and versicolamide B in Aspergillus sp. but has not yet been isolated. The isolation of notoamide S and an enantiomeric mixture of 6-epi-stephacidin A enriched with th...

  11. Impact of isolation method on doubling time and the quality of chondrocyte and osteoblast differentiated from murine dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Rohaya Megat Abdul Wahab

    2017-06-01

    Full Text Available Background Stem cells are normally isolated from dental pulps using the enzymatic digestion or the outgrowth method. However, the effects of the isolation method on the quality of the isolated stem cells are not studied in detail in murine models. The aim of this study was to compare the matrices secreted by osteoblast and chondrocytes differentiated from dental pulp stem cells isolated through different means. Method DPSC from murine incisors were isolated through either the outgrowth (DPSC-OG or the enzymatic digestion (DPSC-ED method. Cells at passage 4 were used in this study. The cells were characterized through morphology and expression of cell surface markers. The cells’ doubling time when cultured using different seeding densities was calculated and analyzed using one-way ANOVA and Tukey’s multiple comparison post-test. The ability of cells to differentiate to chondrocyte and osteoblast was evaluated through staining and analysis on the matrices secreted. Results Gene expression analysis showed that DPSC-OG and DPSC-ED expressed dental pulp mesenchymal stem cell markers, but not hematopoietic stem cell markers. The least number of cells that could have been used to culture DPSC-OG and DPSC-ED with the shortest doubling time was 5 × 102 cells/cm2 (11.49 ± 2.16 h and 1 × 102 cells/cm2 (10.55 h ± 0.50, respectively. Chondrocytes differentiated from DPSC-ED produced  2 times more proteoglycan and at a faster rate than DPSC-OG. FTIR revealed that DPSC-ED differentiated into osteoblast also secreted matrix, which more resembled a calvaria. Discussion Isolation approaches might have influenced the cell populations obtained. This, in turn, resulted in cells with different proliferation and differentiation capability. While both DPSC-OG and DPSC-ED expressed mesenchymal stem cell markers, the percentage of cells carrying each marker might have differed between the two methods. Regardless, enzymatic digestion clearly yielded cells

  12. Toxigenic potentiality of Aspergillus flavus and Aspergillus parasiticus strains isolated from black pepper assessed by an LC-MS/MS based multi-mycotoxin method.

    Science.gov (United States)

    Yogendrarajah, Pratheeba; Devlieghere, Frank; Njumbe Ediage, Emmanuel; Jacxsens, Liesbeth; De Meulenaer, Bruno; De Saeger, Sarah

    2015-12-01

    A liquid chromatography triple quadrupole tandem mass spectrometry method was developed and validated to determine mycotoxins, produced by fungal isolates grown on malt extract agar (MEA). All twenty metabolites produced by different fungal species were extracted using acetonitrile/1% formic acid. The developed method was applied to assess the toxigenic potentiality of Aspergillus flavus (n = 11) and Aspergillus parasiticus (n = 6) strains isolated from black peppers (Piper nigrum L.) following their growth at 22, 30 and 37 °C. Highest mean radial colony growth rates were observed at 30 °C for A. flavus (5.21 ± 0.68 mm/day) and A. parasiticus (4.97 ± 0.33 mm/day). All of the A. flavus isolates produced aflatoxin B1 and O-methyl sterigmatocystin (OMST) while 91% produced aflatoxin B2 (AFB2) and 82% of them produced sterigmatocystin (STERIG) at 30 °C. Except one, all the A. parasiticus isolates produced all the four aflatoxins, STERIG and OMST at 30 °C. Remarkably high AFB1 was produced by some A. flavus isolates at 22 °C (max 16-40 mg/kg). Production of mycotoxins followed a different trend than that of growth rate of both species. Notable correlations were found between different secondary metabolites of both species; R(2) 0.87 between AFB1 and AFB2 production. Occurrence of OMST could be used as a predictor for AFB1 production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. A method for obtaining isolation in production cementing; Sistematica para obtencao de isolamentos nas cimentacoes de producao

    Energy Technology Data Exchange (ETDEWEB)

    Lepeleire, Rubens de; Miletto, Antonio Luis Pires [PETROBRAS, XX (Brazil). Distrito de Perfuracao do Sudeste. Div. Tecnica

    1990-12-31

    This paper presents the procedure used to identify the factors that influenced the appearance of low isolation indexes, 59,1% in production cementing operations in Sao Mateus, during the year of 1987, bringing about a cost increase of over US$200,000.00 in primary cementing corrections. The various stages for the obtainment of such an index are analysed and the modifications that were considered necessary are presented. These modifications raised the index to 91,3% thus reducing correction costs to less than US$150,000.00 in 1988. From January to July, 1989, we able to obtain 98,9% of the isolations and an accumulated cementing correction cost of less than US$20,000.00. (author) 3 refs., 6 figs., 4 tabs.

  14. [Dynamics of the amino acid composition of the medium during cultivation of isolated organs by the directed perfusion method].

    Science.gov (United States)

    Barashkov, V A; Gitel'zon, I I; Nefedov, V P; Trubachev, I N

    1975-07-01

    The dynamics of the amino acid composition of the medium under conditions of adequate perfusion of the isolated organs of a dog (sternum, kidney and liver) was studied. It was found that after a 6-hour perfusion of the complex of organs the amount in the perfusion medium of such amino acids as histidine, lysine, alanine, considerably increased, whereas the amount of arginine, serine, aspartic acid, threonine with glutamine, isoleucine, proline, leucine and valine decreased as compared with their initial concentration. The dynamics of the amino acid medium composition during a 4-hour perfusion was studied in experiments with the isolated sternum. The concentration of alanine, lysine and histidine increased in the medium. At the same time there was seen a decrease in the concentration of serine, aspartic acid, isoleucine, tyrosine and phenyl-alanine.

  15. An optimal method for generating stable vancomycin heteroresistance and intermediate susceptibility in methicillin-resistant Staphylococcus aureusblood isolates under in-vitro vancomycin pressure

    Directory of Open Access Journals (Sweden)

    Riad Khatib

    2012-12-01

    Full Text Available Objectives: The relevance of vancomycin intermediately-susceptible (VISA and hetero-resistant (hVISA methicillin-resistantStaphylococcus aureus (MRSA remains uncertain because of their low frequency. In vitro attempts to generate reducedsusceptibility have been inconsistent. We describe a simple method for generating VISA/hVISA.Materials and methods: Twenty-four SCCmec type II and IV MRSA blood isolates plus USA100 and USA300 controls werecultured (107 CFU/ml in BHI broth with 0, 2 and 3 mg/L vancomycin for 10 days followed by 10 days vancomycin-freepassage.We monitored vancomycin minimum inhibitory concentration (MIC; Etest and population analysis profile-areaunder the curve (PAP-AUC ratios of tested isolate-Mu3 during and after vancomycin pressure (VP. PAP-AUC ratios 1.3 were considered consistent with susceptible, hVISA and VISA, respectively.Results: VP at 2 mg/L (VP-2 increased MIC to 3 mg/L in 20 (83.3% isolates and raised PAP-AUC ratio to hVISA (n=18; 75.0%and VISA (six; 25.0% ranges. VP-3 increased MIC to 3 and 4 mg/L in 19 (79.2% and three (12.5% isolates, respectively andraised PAP-AUC ratio to hVISA (n=3; 12.5% and VISA (n=19; 79.2% ranges. SCCmec IV isolates had lower pre-exposurePAP-AUC ratios (0.48±0.14 vs. 0.69±0.10; p<0.001 and lower MIC (1.6±0.2 vs. 1.8±0.2 mg/L; p=0.1 but MIC rise and VISA/hVISA emergence was comparable. MIC and PAP-AUC ratio rises were stable in 20/22 isolates during drug-free passages.Conclusions: VISA/hVISA readily emerges among MRSA SCCmec type II and IV isolates under VP. VP at the MIC level generateshVISA while pressure at slightly higher level results in the VISA phenotype. J Microbiol Infect Dis 2012; 2(4: 129-134Key words: Staphylococcus aureus; heteroresistance; glycopeptides; resistance; bacteremia.

  16. Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

    Science.gov (United States)

    Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

    2016-09-01

    Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both Phomogenizing sputum samples prior to RNA extraction.

  17. The Antifungal Inhibitory Concentration Effectiveness Test From Ethanol Seed Arabica Coffee (Coffea arabica) Extract Against The Growth Of Candida albicans Patient Isolate With In Vitro Method

    Science.gov (United States)

    Satria Rakatama, Adam; Pramono, Andri; Yulianti, Retno

    2018-03-01

    Candida albicans are the most frequent cause of Vulvovaginalis Candidiasis infection. Its treatment using antifungal drugs, are oftenly caused side effects. The reduction of C.albicans growth and the reduction of antifungal drugs side effect, were our main purposed. Our study objective is determine the effectiveness of inhibitory power of arabica coffee seed ethanol extract on the growth of C.albicans patient isolates. The type of this research is experimental research. Kirby-bauer method with the Saboraud Dextrose Agar (SDA) media was used in this experiment. Inhibitory zone was observed around the disc, to determine the inhibitory power. The results showed that the inhibitory zone was formed on arabica coffee seed ethanol extract on 10%, 20%, 40%, and 80% concentration. Kruskal-Wallis test results (pthe concentration groups tested against the treatment group. The inhibitory zone was formed because of biochemical compound in arabica coffee seed such as caffeine, phenol, alkaloids, flavonoids, and saponins. Inhibitory zone in C.albicans patient isolates were smaller compared with C.albicans ATCC 90028 as gold standard. This showed that the virulence of C.albicans from patients isolates were higher. We concluded that arabica coffee seed ethanol extract could inhibiting the growth of C.albicans patient isolates. Optimization of coffee seed ethanol extract to obtain maximum active ingredients still needs to be done. This knowledge is expected to be used for the beginning manufacturer antifungal drug from natural product.

  18. EPA, DHA, and Lipoic Acid Differentially Modulate the n-3 Fatty Acid Biosynthetic Pathway in Atlantic Salmon Hepatocytes.

    Science.gov (United States)

    Bou, Marta; Østbye, Tone-Kari; Berge, Gerd M; Ruyter, Bente

    2017-03-01

    The aim of the present study was to investigate how EPA, DHA, and lipoic acid (LA) influence the different metabolic steps in the n-3 fatty acid (FA) biosynthetic pathway in hepatocytes from Atlantic salmon fed four dietary levels (0, 0.5, 1.0 and 2.0%) of EPA, DHA or a 1:1 mixture of these FA. The hepatocytes were incubated with [1- 14 C] 18:3n-3 in the presence or absence of LA (0.2 mM). Increased endogenous levels of EPA and/or DHA and LA exposure both led to similar responses in cells with reduced desaturation and elongation of [1- 14 C] 18:3n-3 to 18:4n-3, 20:4n-3, and EPA, in agreement with reduced expression of the Δ6 desaturase gene involved in the first step of conversion. DHA production, on the other hand, was maintained even in groups with high endogenous levels of DHA, possibly due to a more complex regulation of this last step in the n-3 metabolic pathway. Inhibition of the Δ6 desaturase pathway led to increased direct elongation to 20:3n-3 by both DHA and LA. Possibly the route by 20:3n-3 and then Δ8 desaturation to 20:4n-3, bypassing the first Δ6 desaturase step, can partly explain the maintained or even increased levels of DHA production. LA increased DHA production in the phospholipid fraction of hepatocytes isolated from fish fed 0 and 0.5% EPA and/or DHA, indicating that LA has the potential to further increase the production of this health-beneficial FA in fish fed diets with low levels of EPA and/or DHA.

  19. [Identification of anaerobic gram-negative bacilli isolated from various clinical specimens and determination of antibiotic resistance profiles with E-test methods].

    Science.gov (United States)

    Demir, Cengiz; Keşli, Recep

    2018-01-01

    The aim of this study was to identify gram-negative anaerobic bacilli isolated from various clinical specimens that were obtained from patients with suspected anaerobic infections and to determine the antibiotic resistance profiles by using the antibiotic concentration gradient method. The study was performed in Afyon Kocatepe University Ahmet Necdet Sezer Research and Practice Hospital, Medical Microbiology Laboratory between 1 November 2014 and 30 October 2015. Two hundred and seventyeight clinical specimens accepted for anaerobic culture were enrolled in the study. All the samples were cultivated anaerobically by using Schaedler agar with 5% defibrinated sheep blood and Schaedler broth. The isolated anaerobic gram-negative bacilli were identified by using both the conventional methods and automated identification system (VITEK 2, bioMerieux, France). Antibiotic susceptibility tests were performed with antibiotic concentration gradient method (E-test, bioMerieux, France); against penicillin G, clindamycin, cefoxitin, metronidazole, moxifloxacin, imipenem, meropenem, ertapenem and doripenem for each isolate. Of the 28 isolated anaerobic gram-negative bacilli; 14 were identified as Bacteroides fragilis group, 9 were Prevotella spp., and 5 were Fusobacterium spp. The highest resistance rate was found against penicillin (78.5%) and resistance rates against clindamycin and cefoxitin were found as 17.8% and 21.4%, respectively. No resistance was found against metronidazole, moxifloxacin, imipenem, meropenem, ertapenem and doripenem. As a result, isolation and identification of anaerobic bacteria are difficult, time-consuming and more expensive when compared with the cost of aerobic culture. The rate of anaerobic bacteria isolation may be increased by obtaining the appropriate clinical specimen and appropriate transportation of these specimens. We believe that the data obtained from the study in our center may offer benefits for the follow up and treatment of infections

  20. Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk.

    Science.gov (United States)

    Carson, Domonique A; Barkema, Herman W; Naushad, Sohail; De Buck, Jeroen

    2017-09-01

    Non- aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species ( S. capitis , S. chromogenes , S. epidermidis , S. pasteuri , S. saprophyticus , S. sciuri , S. simulans , S. warneri , and S. xylosus ) inhibited growth of S. aureus in vitro , 23 isolates of which, from S. capitis , S. chromogenes , S. epidermidis , S. pasteuri , S. simulans , and S. xylosus , also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus ) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial

  1. Bacteriocins of Non-aureus Staphylococci Isolated from Bovine Milk

    Science.gov (United States)

    Carson, Domonique A.; Barkema, Herman W.; Naushad, Sohail

    2017-01-01

    ABSTRACT Non-aureus staphylococci (NAS), the bacteria most commonly isolated from the bovine udder, potentially protect the udder against infection by major mastitis pathogens due to bacteriocin production. In this study, we determined the inhibitory capability of 441 bovine NAS isolates (comprising 26 species) against bovine Staphylococcus aureus. Furthermore, inhibiting isolates were tested against a human methicillin-resistant S. aureus (MRSA) isolate using a cross-streaking method. We determined the presence of bacteriocin clusters in NAS whole genomes using genome mining tools, BLAST, and comparison of genomes of closely related inhibiting and noninhibiting isolates and determined the genetic organization of any identified bacteriocin biosynthetic gene clusters. Forty isolates from 9 species (S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. saprophyticus, S. sciuri, S. simulans, S. warneri, and S. xylosus) inhibited growth of S. aureus in vitro, 23 isolates of which, from S. capitis, S. chromogenes, S. epidermidis, S. pasteuri, S. simulans, and S. xylosus, also inhibited MRSA. One hundred five putative bacteriocin gene clusters encompassing 6 different classes (lanthipeptides, sactipeptides, lasso peptides, class IIa, class IIc, and class IId) in 95 whole genomes from 16 species were identified. A total of 25 novel bacteriocin precursors were described. In conclusion, NAS from bovine mammary glands are a source of potential bacteriocins, with >21% being possible producers, representing potential for future characterization and prospective clinical applications. IMPORTANCE Mastitis (particularly infections caused by Staphylococcus aureus) costs Canadian dairy producers $400 million/year and is the leading cause of antibiotic use on dairy farms. With increasing antibiotic resistance and regulations regarding use, there is impetus to explore bacteriocins (bacterially produced antimicrobial peptides) for treatment and prevention of bacterial infections

  2. A Simple Response Evaluation Method for Base-Isolation Building-Connection Hybrid Structural System under Long-Period and Long-Duration Ground Motion

    Directory of Open Access Journals (Sweden)

    Kohei Hayashi

    2018-02-01

    Full Text Available An innovative hybrid control building system of base-isolation and building-connection has been proposed in the previous study. This system has two advantages, (i to resist an impulsive earthquake input through the base-isolation system and (ii to withstand a long-duration earthquake input through the building-connection system. A simple response evaluation method without the need of non-linear time–history response analysis is proposed here for this hybrid building system under a long-period and long-duration ground motion. An analytical expression is derived in the plastic deformation of an elastic–perfectly plastic single-degree-of-freedom (SDOF model with viscous damping under the multi-impulse, which is the representative of long-period and long-duration ground motions. A transformation procedure of a base-isolation building-connection hybrid structural system into an SDOF model is proposed by introducing two steps, one is the reduction of the main base-isolated building to an SDOF system, and the other is the reduction of the connecting oil dampers supported on a free-wall to an oil damper with a newly introduced compensation factor on a rigid wall. Application of the analytical expression of the plastic deformation to the reduced SDOF model including the compensation factor on the connecting oil dampers enables the development of a simplified, but rather accurate response evaluation method. The time–history response analysis of the multi-degree-of-freedom model and the comparison with the proposed simplified formula make clear the accuracy and reliability of the proposed simplified response evaluation method.

  3. Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L. leaves

    Directory of Open Access Journals (Sweden)

    Gutha Linga R

    2010-08-01

    Full Text Available Abstract Background Symptoms of grapevine leafroll disease (GLRD in red-fruited wine grape (Vitis vinifera L. cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis. Results We examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR. Using the geNorm program, a combination of two genes (Actin and NAD5 was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3 and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot. The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F3'5'H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus

  4. The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

    Directory of Open Access Journals (Sweden)

    Bo Wu

    2009-07-01

    '-aminolevulinic acid dehydratase (ALAD, the second step. Similarly, Escherichia coli hemH (FC deficient strains transformed with human and Wolbachia FC homologues showed significantly different sensitivities to NMMP. This approach enables functional complementation in E. coli heme deficient mutants as an alternative E. coli-based method for drug screening.Our studies indicate that the heme biosynthetic genes in the Wolbachia of B. malayi (wBm might be essential for the filarial host survival. In addition, the results suggest they are likely candidate drug targets based upon significant differences in phylogenetic distance, biochemical properties and sensitivities to heme biosynthesis inhibitors, as compared to their human homologues.

  5. Open and laparo-endoscopic repair of incarcerated abdominal wall hernias by the use of biological and biosynthetic meshes

    Directory of Open Access Journals (Sweden)

    René H Fortelny

    2016-02-01

    Full Text Available Introduction: Although recently published guidelines recommend against the use of synthetic non-absorbable materials in cases of potentially contaminated or contaminated surgical fields due to the increased risk of infection [1, 2], the use of bio-prosthetic meshes for abdominal wall or ventral hernia repair is still controversially discussed in such cases. Bio-prosthetic meshes have been recommended due to less susceptibility for infection and the decreased risk of subsequent mesh explantation. The purpose of this review is to elucidate if there are any indications for the use of biological and biosynthetic meshes in incarcerated abdominal wall hernias based on the recently published literature.Methods: A literature search of the Medline database using the PubMed search engine, using the keywords returned 486 articles up to June 2015. The full text of 486 articles was assessed and 13 relevant papers were identified including 5 retrospective case cohort studies, 2 case controlled studies, 6 case series.Results: The results of Franklin et al [23, 24, 25] included the highest number of biological mesh repairs (Surgisis® by laparoscopic IPOM in infected fields which demonstrated a very low incidence of infection and recurrence (0,7% and 5,2%. Han et al [26] reported in his retrospective study the highest number of treated patients due to incarcerated hernias by open approach using acellular dermal matrix (ADM® with very low rate of infection as well as recurrences (1,6% and 15,9. Both studies achieved acceptable outcome in a follow up of at least 3,5 years compared to the use of synthetic mesh in this high-risk population [3]Conclusion:Currently there is a very limited evidence for the use of biological and biosynthetic meshes in strangulated hernias in either open or laparo-endoscopic repair. Finally, there is an urgent need to start with randomized controlled comparative trials as well as to support registries with data to achieve more

  6. De novo transcriptome sequencing and digital gene expression analysis predict biosynthetic pathway of rhynchophylline and isorhynchophylline from Uncaria rhynchophylla, a non-model plant with potent anti-alzheimer's properties.

    Science.gov (United States)

    Guo, Qianqian; Ma, Xiaojun; Wei, Shugen; Qiu, Deyou; Wilson, Iain W; Wu, Peng; Tang, Qi; Liu, Lijun; Dong, Shoukun; Zu, Wei

    2014-08-12

    The major medicinal alkaloids isolated from Uncaria rhynchophylla (gouteng in chinese) capsules are rhynchophylline (RIN) and isorhynchophylline (IRN). Extracts containing these terpene indole alkaloids (TIAs) can inhibit the formation and destabilize preformed fibrils of amyloid β protein (a pathological marker of Alzheimer's disease), and have been shown to improve the cognitive function of mice with Alzheimer-like symptoms. The biosynthetic pathways of RIN and IRN are largely unknown. In this study, RNA-sequencing of pooled Uncaria capsules RNA samples taken at three developmental stages that accumulate different amount of RIN and IRN was performed. More than 50 million high-quality reads from a cDNA library were generated and de novo assembled. Sequences for all of the known enzymes involved in TIAs synthesis were identified. Additionally, 193 cytochrome P450 (CYP450), 280 methyltransferase and 144 isomerase genes were identified, that are potential candidates for enzymes involved in RIN and IRN synthesis. Digital gene expression profile (DGE) analysis was performed on the three capsule developmental stages, and based on genes possessing expression profiles consistent with RIN and IRN levels; four CYP450s, three methyltransferases and three isomerases were identified as the candidates most likely to be involved in the later steps of RIN and IRN biosynthesis. A combination of de novo transcriptome assembly and DGE analysis was shown to be a powerful method for identifying genes encoding enzymes potentially involved in the biosynthesis of important secondary metabolites in a non-model plant. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the capsule extract from Uncaria, and provides information that may aid in metabolic engineering to increase yields of these important alkaloids.

  7. The discrimination of d-tartrate positive and d-tartrate negative S. enterica subsp. enterica serovar Paratyphi B isolated in Malaysia by phenotypic and genotypic methods.

    Science.gov (United States)

    Ahmad, Norazah; Hoon, Shirley Tang Gee; Ghani, Mohamed Kamel Abd; Tee, Koh Yin

    2012-06-01

    Serotyping is not sufficient to differentiate between Salmonella species that cause paratyphoid fever from the strains that cause milder gastroenteritis as these organisms share the same serotype Salmonella Paratyphi B (S. Paratyphi B). Strains causing paratyphoid fever do not ferment d-tartrate and this key feature was used in this study to determine the prevalence of these strains among the collection of S. Paratyphi B strains isolated from patients in Malaysia. A total of 105 isolates of S. Paratyphi B were discriminated into d-tartrate positive (dT+) and d-tartrate negative (dT) variants by two lead acetate test protocols and multiplex PCR. The lead acetate test protocol 1 differed from protocol 2 by a lower inoculum size and different incubation conditions while the multiplex PCR utilized 2 sets of primers targeting the ATG start codon of the gene STM3356. Lead acetate protocol 1 discriminated 97.1% of the isolates as S. Paratyphi B dT+ and 2.9% as dT while test protocol 2 discriminated all the isolates as S. Paratyphi B dT+. The multiplex PCR test identified all 105 isolates as S. Paratyphi B dT+ strains. The concordance of the lead acetate test relative to that of multiplex PCR was 97.7% and 100% for protocol 1 and 2 respectively. This study showed that S. Paratyphi B dT+ is a common causative agent of gastroenteritis in Malaysia while paratyphoid fever appears to be relatively uncommon. Multiplex PCR was shown to be a simpler, more rapid and reliable method to discriminate S. Paratyphi B than the phenotypic lead acetate test.

  8. Development of a gene cloning system in a fast-growing and moderately thermophilic Streptomyces species and heterologous expression of Streptomyces antibiotic biosynthetic gene clusters

    Science.gov (United States)

    2011-01-01

    Background Streptomyces species are a major source of antibiotics. They usually grow slowly at their optimal temperature and fermentation of industrial strains in a large scale often takes a long time, consuming more energy and materials than some other bacterial industrial strains (e.g., E. coli and Bacillus). Most thermophilic Streptomyces species grow fast, but no gene cloning systems have been developed in such strains. Results We report here the isolation of 41 fast-growing (about twice the rate of S. coelicolor), moderately thermophilic (growing at both 30°C and 50°C) Streptomyces strains, detection of one linear and three circular plasmids in them, and sequencing of a 6996-bp plasmid, pTSC1, from one of them. pTSC1-derived pCWH1 could replicate in both thermophilic and mesophilic Streptomyces strains. On the other hand, several Streptomyces replicons function in thermophilic Streptomyces species. By examining ten well-sporulating strains, we found two promising cloning hosts, 2C and 4F. A gene cloning system was established by using the two strains. The actinorhodin and anthramycin biosynthetic gene clusters from mesophilic S. coelicolor A3(2) and thermophilic S. refuineus were heterologously expressed in one of the hosts. Conclusions We have developed a gene cloning and expression system in a fast-growing and moderately thermophilic Streptomyces species. Although just a few plasmids and one antibiotic biosynthetic gene cluster from mesophilic Streptomyces were successfully expressed in thermophilic Streptomyces species, we expect that by utilizing thermophilic Streptomyces-specific promoters, more genes and especially antibiotic genes clusters of mesophilic Streptomyces should be heterologously expressed. PMID:22032628

  9. In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in Parsley (Petroselinum crispum)

    International Nuclear Information System (INIS)

    Reinold, S.; Hahlbrock, K.

    1997-01-01

    Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together constituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously est