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Sample records for biosynthetic methods isolation

  1. High-Yield Method for Isolation and Culture of Endothelial Cells from Rat Coronary Blood Vessels Suitable for Analysis of Intracellular Calcium and Nitric Oxide Biosynthetic Pathways

    Science.gov (United States)

    Nistri, Silvia; Mazzetti, Luca; Failli, Paola

    2002-01-01

    We describe here a method for isolating endothelial cells from rat heart blood vessels by means of coronary microperfusion with collagenase. This methods makes it possible to obtain high amounts of endothelial cells in culture which retain the functional properties of their in vivo counterparts, including the ability to uptake fluorescently-labeled acetylated low-density lipoproteins and to respond to vasoactive agents by modulating intracellular calcium and by upregulating intrinsic nitric oxide generation. The main advantages of our technique are: (i) good reproducibility, (ii) accurate sterility that can be maintained throughout the isolation procedure and (iii) high yield of pure endothelial cells, mainly due to microperfusion and temperature-controlled incubation with collagenase which allow an optimal distribution of this enzyme within the coronary vascular bed. PMID:12734571

  2. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gammeltoft, S.

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-/sup 125/I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity.

  3. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    International Nuclear Information System (INIS)

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-125I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity

  4. 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.

  5. 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)

    A method is described for the preparation and analysis of adenylic, uri dilic, cytidi- 11c and guanylic acids, labelled with 14C. Escherichia coli cells have been labelled by growing them in a medi dia containing glucose-14C 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

  6. Nanolipoprotein particles comprising a natural rubber biosynthetic enzyme complex and related products, methods and systems

    Energy Technology Data Exchange (ETDEWEB)

    Hoeprich, Paul D.; Whalen, Maureen

    2016-04-05

    Provided herein are nanolipoprotein particles that comprise a biosynthetic enzyme more particularly an enzyme capable of catalyzing rubber or other rubbers polymerization, and related assemblies, devices, methods and systems.

  7. Screening for the presence of biosynthetic genes for antimicrobial lipopeptides in natural isolates of Bacillus sp.

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    Stanković S.

    2012-01-01

    Full Text Available A collection of 205 natural isolates of Bacillus was tested for the presence of genes for biosynthesis of antimicrobial lipopeptides, iturin, surfactin, fengycin and bacillomycin D. For the detection of iturin producers by PCR screening, we used forward ITUP1-F and reverse ITUP2-R primers which are capable of detecting a 2-kb region that includes the intergenic sequence between the ituA and ituB genes. A 675-bp fragment from the gene sfp from B. subtilis encoding 4’-phosphopantetheinyl transferase involved in the biosynthesis of surfactin was targeted for amplification by using primers P17 and P18. Other two pairs of primers were BACC1F and BACC1R for bacillomycin D and FEND1F and FEND1R for potential fengycin producers, respectively. The results of the screening showed that the majority of tested strains had more than one biosynthetic operon, since 81% possessed the genes for bacillomycin D production, 54% for surfactin, 38% for iturin and 25% for fengycin production. [Projekat Ministarstva nauke Republike Srbije, br. 173026

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

    OpenAIRE

    Zuiter Afnan; Sawwan Jammal; Al Abdallat Ayed

    2012-01-01

    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...

  9. Diversity and biosynthetic potential of culturable aerobic heterotrophic bacteria isolated from Magura Cave, Bulgaria

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    Tomova Iva

    2013-01-01

    Full Text Available Biocapacity of bacteria inhabiting karstic caves to produce valuable biologically active compounds is still slightly investigated. A total of 46 culturable heterotrophic bacteria were isolated under aerobic conditions from the Gallery with pre-historical drawings in Magura Cave, Bulgaria. Phylogenetic analysis revealed that most of bacterial isolates aff iliated with Proteobacteria (63%, followed by Actinobacteria (10.9%, Bacteroidetes (10.9%, and Firmicutes (6.5%. A strong domination of Gram-negative bacteria (total 81% belonging to nine genera: Serratia, Pseudomonas, Enterobacter, Sphingobacterium, Stenotrophomonas, Commamonas, Acinetobacter, Obesumbacterium, and Myroides, was observed. Gram-positive isolates were represented by the genera Bacillus, Arthrobacter, and Micrococcus. One isolate showed a signif icant phylogenetic distance to the closest neighbor and could represent а novel species. Heterotrophic bacterial isolates from Magura Cave were investigated for hydrolytic enzymes production, antimicrobial and hemolytic activity. Predominance of producers of protease (87%, followed by xanthan lyase (64%, lipase (40%, β-glycosidase (40%, and phytase (21% was observed. Over 75% of the isolates demonstrated antimicrobial and hemolytic activity. The results suggest that heterotrophic bacteria isolated from Magura Cave could be a valuable source of industrially relevant psychrotolerant enzymes and bioactive metabolites. This study is a f irst report on the taxonomic composition and biological activity of culturable bacteria inhabiting a cave in Bulgaria.

  10. Pyocyanine Biosynthetic Genes in Clinical and Environmental Isolates of Pseudomonas aeruginosa and Detection of Pyocyanine’s Antimicrobial Effects with or without Colloidal Silver Nanoparticles

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    Afrooz Rashnonejad

    2012-01-01

    Full Text Available Objective: Pyocyanine plays an important role in the pathogenesis of Pseudomonas aeruginosa, (P. aeruginosa and is known to have inhibitory and bactericidal effects. This study has aimed to detect the phenazine biosynthetic operon (phz ABCDEFG and two phenazine modifying genes (phzM and phzS by polymerase chain reaction (PCR and detection of its possible protein bands by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE. The antimicrobial effects of pyocyanine alone and mixed with colloidal silver nanoparticles were studied.Materials and Methods: In this descriptive study, clinical and environmental species of P. aeruginosa were isolated by thioglycollate medium culture and cetrimide agar, respectively. The existence of a phenazine biosynthetic operon and two phenazine modifying genes as well as their protein products were confirmed by PCR and SDS-PAGE, respectively. Pyocyanine was extracted with chloroform and its antimicrobial effects against bacteria such as; Escherichia coli (E. coli, P. aeruginosaand Staphylococcus aureus (S. aureus bacteria and yeast Candida albicans (C. albicans were tested using well, spot and disk diffusion methods.Results: In this study, 3 out of 48 clinical strains were unable to produce pyocyanine on cetrimide and Mueller Hinton (MH agar. Two strains did not have phenazine modifying gene bands. Another strain did not have the possible protein band of the phzM gene. Pyocyanine had antimicrobial effects against the microbial strains, which increased in the presence of silver nanoparticles.Conclusion: According to the results of the present study, some P. aeruginosa strains are unable to produce pyocyanine due to the absence of the phzM or phzS genes. Therefore, these genes have an important role in pyocyanine production in P. aeruginosa. Pyocyanine shows synergistic antimicrobial effects in the presence of silver nanoparticles against microbial strains.

  11. Method for isolating nucleic acids

    Science.gov (United States)

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  12. Rifamycin Biosynthetic Congeners: Isolation and Total Synthesis of Rifsaliniketal and Total Synthesis of Salinisporamycin and Saliniketals A and B.

    Science.gov (United States)

    Feng, Yu; Liu, Jun; Carrasco, Yazmin P; MacMillan, John B; De Brabander, Jef K

    2016-06-01

    We describe the isolation, structure elucidation, and total synthesis of the novel marine natural product rifsaliniketal and the total synthesis of the structurally related variants salinisporamycin and saliniketals A and B. Rifsaliniketal was previously proposed, but not observed, as a diverted metabolite from a biosynthetic precursor to rifamycin S. Decarboxylation of rifamycin provides salinisporamycin, which upon truncation with loss of the naphthoquinone ring leads to saliniketals. Our synthetic strategy hinged upon a Pt(II)-catalyzed cycloisomerization of an alkynediol to set the dioxabicyclo[3.2.1]octane ring system and a fragmentation of an intermediate dihydropyranone to forge a stereochemically defined (E,Z)-dienamide unit. Multiple routes were explored to assemble fragments with high stereocontrol, an exercise that provided additional insights into acyclic stereocontrol during stereochemically complex fragment-assembly processes. The resulting 11-14 step synthesis of saliniketals then enabled us to explore strategies for the synthesis and coupling of highly substituted naphthoquinones or the corresponding naphthalene fragments. Whereas direct coupling with naphthoquinone fragments proved unsuccessful, both amidation and C-N bond formation tactics with the more electron-rich naphthalene congeners provided an efficient means to complete the first total synthesis of rifsaliniketal and salinisporamycin. PMID:27232659

  13. Screening for the presence of biosynthetic genes for antimicrobial lipopeptides in natural isolates of Bacillus sp.

    OpenAIRE

    Stanković S.; Mihajlović Sanja; Draganić V.; Dimkić I.; Vukotić G.; Berić Tanja; Fira Đ.

    2012-01-01

    A collection of 205 natural isolates of Bacillus was tested for the presence of genes for biosynthesis of antimicrobial lipopeptides, iturin, surfactin, fengycin and bacillomycin D. For the detection of iturin producers by PCR screening, we used forward ITUP1-F and reverse ITUP2-R primers which are capable of detecting a 2-kb region that includes the intergenic sequence between the ituA and ituB genes. A 675-bp fragment from the gene sfp from B. subtilis encoding 4’-phosphopantetheinyl ...

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. Isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential.

    Science.gov (United States)

    Passari, Ajit K; Mishra, Vineet K; Saikia, Ratul; Gupta, Vijai K; Singh, Bhim P

    2015-01-01

    Microorganisms associated with medicinal plants are of interest as the producers of important bioactive compounds. To date, the diversity of culturable endophytic actinomycetes associated with medicinal plants is in its initial phase of exploration. In this study, 42 endophytic actinomycetes were isolated from different organs of seven selected medicinal plants. The highest number of isolates (n = 22, 52.3%) of actinomycetes was isolated from roots, followed by stems (n = 9, 21.4%), leaves (n = 6, 14.2%), flowers (n = 3, 7.1%), and petioles (n = 2, 4.7%). The genus Streptomyces was the most dominant among the isolates (66.6%) in both the locations (Dampa TRF and Phawngpuii NP, Mizoram, India). From a total of 42 isolates, 22 isolates were selected for further studies based on their ability to inhibit one of the tested human bacterial or fungal pathogen. Selected isolates were identified based on 16S rRNA gene analysis and subsequently the isolates were grouped to four different genera; Streptomyces, Brevibacterium, Microbacterium, and Leifsonia. Antibiotic sensitivity assay was performed to understand the responsible antimicrobials present in the isolates showing the antimicrobial activities and revealed that the isolates were mostly resistant to penicillin G and ampicillin. Further, antimicrobial properties and antibiotic sensitivity assay in combination with the results of amplification of biosynthetic genes polyketide synthase (PKS-I) and non-ribosomal peptide synthetase (NRPS) showed that the endophytic actinomycetes associated with the selected medicinal plants have broad-spectrum antimicrobial activity. This is the first report of the isolation of Brevibacterium sp., Microbacterium sp., and Leifsonia xyli from endophytic environments of medicinal plants, Mirabilis jalapa and Clerodendrum colebrookianum. Our results emphasize that endophytic actinomycetes associated with medicinal plants are an unexplored resource for the discovery of biologically active

  19. Isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential

    Directory of Open Access Journals (Sweden)

    Ajit Kumar Passari

    2015-04-01

    Full Text Available Microorganisms associated with medicinal plants are of interest as the producers of important bioactive compounds. To date, the diversity of culturable endophytic actinomycetes associated with medicinal plants is in its initial phase of exploration. In this study, 42 endophytic actinomycetes were isolated from different organs of seven selected medicinal plants. The highest number of isolates (n=22, 52.3% of actinomycetes was isolated from roots, followed by stems (n=9, 21.4%, leaves (n=6, 14.2%, flowers (n=3, 7.1% and petioles (n=2, 4.7%. The genus Streptomyces was the most dominant among the isolates (66.6% in both the locations (Dampa TRF and Phawngpuii NP, Mizoram, India. From a total of 42 isolates, 22 isolates were selected for further studies based on their ability to inhibit one of the tested human bacterial or fungal pathogen. Selected isolates were identified based on 16S rRNA gene analysis and subsequently the isolates were grouped to four different genera; Streptomyces, Brevibacterium, Microbacterium and Leifsonia. Antibiotic sensitivity assay was performed to understand the responsible antimicrobials present in the isolates showing the antimicrobial activities and revealed that the isolates were mostly resistant to penicillin G and ampicillin. Further, antimicrobial properties and antibiotic sensitivity assay in combination with the results of amplification of biosynthetic genes polyketide synthase (PKS-I and nonribosomal peptide synthetase (NRPS showed that the endophytic actinomycetes associated with the selected medicinal plants have broad-spectrum antimicrobial activity. This is the first report of the isolation of Brevibacterium sp., Microbacterium sp. and Leifsonia xyli from endophytic environments of medicinal plants, Mirabilis jalapa and Clerodendrum colebrookianum. Our results emphasize that endophytic actinomycetes associated with medicinal plants are an unexplored resource for the discovery of biologically active

  20. Identification and characterization of a new erythromycin biosynthetic gene cluster in Actinopolyspora erythraea YIM90600, a novel erythronolide-producing halophilic actinomycete isolated from salt field.

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    Dandan Chen

    Full Text Available Erythromycins (Ers are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by Saccharopolyspora erythraea, are mainly soil-dwelling actinomycetes. So far, Actinopolyspora erythraea YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of Ac. erythraea YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of Sa. erythraea NRRL2338, except for two absent genes, eryBI and eryG. By correlating genotype and chemotype, the biosynthetic pathways of 3'-demethyl-erythromycin C, erythronolide H (EH and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an in vitro enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryFAc (derived from Ac. erythraea YIM90600 in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing Sa. erythraea mutant via gene complementation was not successful. Characterization of EryKAc (derived from Ac. erythraea YIM90600 in vitro has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile Ac. erythraea YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.

  1. Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis

    OpenAIRE

    Nannemann, David P.; Birmingham, William R.; Scism, Robert A.; Bachmann, Brian O.

    2011-01-01

    To address the synthesis of increasingly structurally diverse small-molecule drugs, methods for the generation of efficient and selective biological catalysts are becoming increasingly important. ‘Directed evolution’ is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaign...

  2. Identification of Quorum-Sensing Signal Molecules and a Biosynthetic Gene in Alicycliphilus sp. Isolated from Activated Sludge.

    Science.gov (United States)

    Morohoshi, Tomohiro; Okutsu, Noriya; Xie, Xiaonan; Ikeda, Tsukasa

    2016-01-01

    Activated sludge is a complicated mixture of various microorganisms that is used to treat sewage and industrial wastewater. Many bacteria produce N-acylhomoserine lactone (AHL) as a quorum-sensing signal molecule to regulate the expression of the exoenzymes used for wastewater treatment. Here, we isolated an AHL-producing bacteria from an activated sludge sample collected from an electronic component factory, which we named Alicycliphilus sp. B1. Clone library analysis revealed that Alicycliphilus was a subdominant genus in this sample. When we screened the activated sludge sample for AHL-producing strains, 12 of 14 the AHL-producing isolates were assigned to the genus Alicycliphilus. A putative AHL-synthase gene, ALISP_0667, was cloned from the genome of B1 and transformed into Escherichia coli DH5α. The AHLs were extracted from the culture supernatants of the B1 strain and E. coli DH5α cells harboring the ALISP_0667 gene and were identified by liquid chromatography-mass spectrometry as N-(3-hydroxydecanoyl)-l-homoserine lactone and N-(3-hydroxydodecanoyl)-l-homoserine lactone. The results of comparative genomic analysis suggested that the quorum-sensing genes in the B1 strain might have been acquired by horizontal gene transfer within activated sludge. PMID:27490553

  3. 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. PMID:26348620

  4. Method development and analysis of free HS and HS in proteoglycans from pre- and postmenopausal women: Evidence for biosynthetic pathway changes in sulfotransferase and sulfatase enzymes

    Science.gov (United States)

    Wei, Wei; Miller, Rebecca L.; Leary, Julie A.

    2013-01-01

    Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis, however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration and solid phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated. PMID:23659730

  5. Development of analysis methods for seismically isolated nuclear structures

    International Nuclear Information System (INIS)

    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)

  6. Introducing Organic Chemistry Students to Natural Product Isolation Using Steam Distillation and Liquid Phase Extraction of Thymol, Camphor, and Citral, Monoterpenes Sharing a Unified Biosynthetic Precursor

    Science.gov (United States)

    McLain, Katherine A.; Miller, Kenneth A.; Collins, William R.

    2015-01-01

    Plants have provided and continue to provide the inspiration and foundation for modern medicines. Natural product isolation is a key component of the process of drug discovery from plants. The purpose of this experiment is to introduce first semester undergraduate organic chemistry students, who have relatively few lab techniques at their…

  7. Biosynthetic origin of acetic acid using SNIF-NMR

    International Nuclear Information System (INIS)

    The main purpose of this work is to describe the use of the technique Site-Specific Natural Isotopic Fractionation of hydrogen (SNIF-NMR), using 2H and 1H 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 C3, C4, and CAM biosynthetic mechanisms, blends of C3 and C4 (agrins) and synthetic acetic acid. (author)

  8. Novel method for selective isolation of actinomycetes.

    OpenAIRE

    Hirsch, C F; Christensen, D L

    1983-01-01

    A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed...

  9. Design and application of the method for isolating magnetotactic bacteria

    Institute of Scientific and Technical Information of China (English)

    XIAO Zhijie; LIAN Bin; CHEN Jun; H. Henry Teng

    2007-01-01

    A simple apparatus was designed to effectively isolate magnetotactic bacteria from soils or sediments based on their magnetotaxis. Through a series of processes including sample incubation, MTB harvesting, isolation, purification and identification, several strains of bacteria were isolated from the samples successfully. By Transmission Electron Microscopy (TEM) and Energy-Dispersive X-ray Analysis (EDXA), these bacteria were certificated to be magnetotactic bacteria. The phylogenetic relationship between the isolated magnetic strains and some known magnetotactic bacteria was inferred by the construction of phylogenetic tree based on 16SrDNA sequences. This apparatus has been proven to have the advantages of being inexpensive, simple to assemble, easy to perform and highly efficient to isolate novel magnetotactic bacteria. The research indicated that the combined approach of harvesting MTB by home-made apparatus and the method of plate colony isolation could purify and isolate magnetotactic bacteria effectively.

  10. Biosynthetic engineering of nonribosomal peptide synthetases.

    Science.gov (United States)

    Kries, Hajo

    2016-09-01

    From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27465074

  11. Improved Method for Isolation of Bacterial Inhibitors from Oleuropein Hydrolysis

    OpenAIRE

    Federici, Federico; Bongi, Guido

    1983-01-01

    A new high-pressure liquid chromatography multidetection quantitative method for the isolation of the products of oleuropein hydrolysis is described. A single analysis yields sufficient amounts of the compounds to test their inhibitory effect on bacterial growth.

  12. 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

  13. A Novel Method Isolated Microorganisms in Soil Granule

    Institute of Scientific and Technical Information of China (English)

    Liu Bao-ping; Xiang Wen-sheng; Wang Hong-yan; Fu Shi-cong

    2012-01-01

    A novel method isolated microorganisms in soil granule was built. The key steps included: repeated elutriation of soil by sterilized water, inoculation on the plates with the elutriated sediments, incubation of the plates and isolation of the actinomycetes by using selected culture medium. We formulated that most microflora included the dominant actinomycetes in the soil were carried away with the sterilized water in the elutriation procedure, some rare actinomycetes and few other microflora included bacteria were remained in the elutriated sediments, the other microflora were excluded to grew into colonies on the plates by using selective culture medium for actinomycetes in the elutriated sediments. Results showed the supposition. Non-streptomycete actinomycetes were isolated both from black soil samples from Chinese northeast area and compost samples from Chinese central area. Soil fungi in granule were isolated by using the selective conditions to favor fungi. The results showed that the method was effective

  14. 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

  15. A Damping Characteristics Calculation Method of Metal Dry Friction Isolators

    Institute of Scientific and Technical Information of China (English)

    JIANG Hong-yuan; HAO De-gang; XIA Yu-hong; ULANOV A M; PONOMAREV Yu K

    2008-01-01

    The dry friction ring-type vibration isolator is considered as an isotropic continuous medium. A method of dry friction hysteresis loop calculation is proposed based on friction force analysis of contact beam. The friction force is modeled as an equivalent distributed moment to use the finite element method (FEM) to calculate the dry friction vibration isolator hysteresis loop, so the damping characteristics can be obtained. A comparison of the hysteresis loop calculation results and the experimental results shows the average relative error is 2.7%, it proves the calculation method is feasible.

  16. An automatic and effective tooth isolation method for dental radiographs

    Science.gov (United States)

    Lin, P.-L.; Huang, P.-W.; Cho, Y. S.; Kuo, C.-H.

    2013-03-01

    Tooth isolation is a very important step for both computer-aided dental diagnosis and automatic dental identification systems, because it will directly affect the accuracy of feature extraction and, thereby, the final results of both types of systems. This paper presents an effective and fully automatic tooth isolation method for dental X-ray images, which contains up-per-lower jaw separation, single tooth isolation, over-segmentation verification, and under-segmentation detection. The upper-lower jaw separation mechanism is based on a gray-scale integral projection to avoid possible information loss and incorporates with the angle adjustment to handle skewed images. In a single tooth isolation, an adaptive windowing scheme for locating gap valleys is proposed to improve the accuracy. In over-segmentation, an isolation-curve verification scheme is proposed to remove excessive curves; and in under-segmentation, a missing-teeth detection scheme is proposed. The experimental results demonstrate that our method achieves the accuracy rates of 95.63% and 98.71% for the upper and lower jaw images, respectively, from the test database of 60 bitewing dental radiographs, and performs better for images with severe teeth occlusion, excessive dental works, and uneven illumination than that of Nomir and Abdel-Mottaleb's method. The method without upper-lower jaw separation step also works well for panoramic and periapical images.

  17. A convenient method for saponin isolation in tumour therapy.

    Science.gov (United States)

    Weng, Alexander; Jenett-Siems, Kristina; Schmieder, Peter; Bachran, Diana; Bachran, Christopher; Görick, Cornelia; Thakur, Mayank; Fuchs, Hendrik; Melzig, Matthias F

    2010-03-01

    Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy. PMID:20144565

  18. Recycling Isolation of Plant DNA, A Novel Method

    Institute of Scientific and Technical Information of China (English)

    Lingling Zhang; Bo Wang; Lei Pan; Junhua Peng

    2013-01-01

    DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and goodquality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations (termed as the 1st,2nd,3rd and 4th DNA sample,respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.

  19. Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria.

    Science.gov (United States)

    Blodgett, Joshua A V; Oh, Dong-Chan; Cao, Shugeng; Currie, Cameron R; Kolter, Roberto; Clardy, Jon

    2010-06-29

    A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identification of the frontalamide biosynthetic gene cluster and several biosynthetic intermediates. The biosynthetic locus for the frontalamides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), which resembles iterative enzymes known in fungi. No such mixed iterative PKS-NRPS enzymes have been characterized in bacteria. Genome-mining efforts revealed strikingly conserved frontalamide-like biosynthetic clusters in the genomes of phylogenetically diverse bacteria ranging from proteobacteria to actinomycetes. Screens for environmental actinomycete isolates carrying frontalamide-like biosynthetic loci led to the isolation of a number of positive strains, the majority of which produced candidate frontalamide-like compounds under suitable growth conditions. These results establish the prevalence of frontalamide-like gene clusters in diverse bacterial types, with medicinally important Streptomyces species being particularly enriched. PMID:20547882

  20. A microRNA isolation method from clinical samples

    Science.gov (United States)

    Zununi Vahed, Sepideh; Barzegari, Abolfazl; Rahbar Saadat, Yalda; Mohammadi, Somayeh; Samadi, Nasser

    2016-01-01

    Introduction: microRNAs (miRNAs) are considered to be novel molecular biomakers that could be exploited in the diagnosis and treatment of different diseases. The present study aimed to develop an efficient miRNA isolation method from different clinical specimens. Methods: Total RNAs were isolated by Trizol reagent followed by precipitation of the large RNAs with potassium acetate (KCH3COOH), polyethylene glycol (PEG) 4000 and 6000, and lithium chloride (LiCl). Then, small RNAs were enriched and recovered from the supernatants by applying a combination of LiCl and ethanol. The efficiency of the method was evaluated through the quality, quantity, and integrity of the recovered RNAs using the A260/280 absorbance ratio, reverse transcription PCR (RT-PCR), and quantitative real-time PCR (q-PCR). Results: Comparison of different RNA isolation methods based on the precipitation of DNA and large RNAs, high miRNA recovery and PCR efficiency revealed that applying potassium acetate with final precipitation of small RNAs using 2.5 M LiCl plus ethanol can provide high yield and quality small RNAs that can be exploited for clinical purposes. Conclusion: The current isolation method can be applied for most clinical samples including cells, formalin-fixed and paraffin-embedded (FFPE) tissues and even body fluids with a wide applicability in molecular biology investigations.

  1. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    OpenAIRE

    Mie Bech Lukassen; Wagma Saei; Teis Esben Sondergaard; Anu Tamminen; Abhishek Kumar; Frank Kempken; Wiebe, Marilyn G.; Jens Laurids Sørensen

    2015-01-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus n...

  2. Biosynthetic origin of mycobacterial cell wall arabinosyl residues.

    OpenAIRE

    Scherman, M.; Weston, A; Duncan, K; Whittington, A; Upton, R; Deng, L.; Comber, R; Friedrich, J D; McNeil, M

    1995-01-01

    Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14C or 3H in specific positions. The resulting radiolabeled cell walls were isolated and hydrolyzed, ...

  3. Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system

    OpenAIRE

    Nah, Hee-Ju; Woo, Min-Woo; Choi, Si-Sun; Kim, Eung-Soo

    2015-01-01

    Background Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-si...

  4. New Rapid Method of DNA Isolation from Milk Somatic Cells.

    Science.gov (United States)

    Pokorska, Joanna; Kułaj, Dominika; Dusza, Magdalena; Żychlińska-Buczek, Justyna; Makulska, Joanna

    2016-04-01

    Isolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses. PMID:26913552

  5. Optical method of recording electrical activity in isolated rabbit hearts

    OpenAIRE

    Amanna, Ashwin E

    1993-01-01

    A recently developed optical method utilizes a single, implantable, optical fiber to record electrical activity from isolated hearts stained with voltage-sensitive dyes. This optical technique generates recordings of transmembrane potential from excitable myocardial tissue, and remain free from stimulus artifacts that accompany electro stimulation and hinder all standard electrode recording methods during the application of high-voltage electrical shocks. The fiber optic system...

  6. Current methods for the isolation of extracellular vesicles.

    Science.gov (United States)

    Momen-Heravi, Fatemeh; Balaj, Leonora; Alian, Sara; Mantel, Pierre-Yves; Halleck, Allison E; Trachtenberg, Alexander J; Soria, Cesar E; Oquin, Shanice; Bonebreak, Christina M; Saracoglu, Elif; Skog, Johan; Kuo, Winston Patrick

    2013-10-01

    Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter. PMID:23770532

  7. Rapid and inexpensive method for isolating plasmid DNA

    International Nuclear Information System (INIS)

    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

  8. 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.

  9. Method for strontium isolation from high-mineralized water

    International Nuclear Information System (INIS)

    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 SrCO3 constitutes 90.5% of its content in the initial solution. The method presented can be used for strontium separation from natural and waste waters

  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. A Method to Isolate Viable Schwann Cells from Adult Rat

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 Introduction Schwann cells (SCs) are the glial cells of the peripheral nervous system, which play an important role for repairing nerve injuries and demyelination diseases. The ability to generate large numbers of viable SCs in a short period of time from adult peripheral nerves makes them potential candidates for the clinical application of cell transplantation to enhance remyelination in human demyelinating disease and repair nerve damage. Previously most methods to isolate SCs are not clinically accept...

  12. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Science.gov (United States)

    Lukassen, Mie Bech; Saei, Wagma; Sondergaard, Teis Esben; Tamminen, Anu; Kumar, Abhishek; Kempken, Frank; Wiebe, Marilyn G.; Sørensen, Jens Laurids

    2015-01-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster. PMID:26184239

  13. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Directory of Open Access Journals (Sweden)

    Mie Bech Lukassen

    2015-07-01

    Full Text Available Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine. Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1, a polyketide synthase (PKS2, a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

  14. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis.

    Science.gov (United States)

    Lukassen, Mie Bech; Saei, Wagma; Sondergaard, Teis Esben; Tamminen, Anu; Kumar, Abhishek; Kempken, Frank; Wiebe, Marilyn G; Sørensen, Jens Laurids

    2015-07-01

    Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl) attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine). Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1), a polyketide synthase (PKS2), a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster. PMID:26184239

  15. 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....

  16. 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

  17. ADVANCED SEISMIC BASE ISOLATION METHODS FOR MODULAR REACTORS

    International Nuclear Information System (INIS)

    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

  18. Simple method of isolating humic acids from organic soils

    Science.gov (United States)

    Ahmed, O. H.; Susilawati, K.; Nik Muhamad, A. B.; Khanif, M. Y.

    2009-04-01

    Humic substances particularly humic acids (HA) play a major role in soil conditioning e.g. erosion control, soil cation exchange capacity, complexation of heavy metal ions and pesticides, carbon and nitrogen cycles, plant growth and reduction of ammonia volatilization from urea. Humified substances such as coal, composts, and peat soils have substantial amounts of HA but the isolation of these acids is expensive, laborious, and time consuming. Factors that affect the quality and yield of HA isolated from these materials include extraction, fractionation, and purification periods. This work developed a simple, rapid, and cost effective method of isolating HA from peat soils. There was a quadratic relationship between extraction period and HA yield. Optimum extraction period was estimated at 4 h instead of the usual range of 12 to 48 h. There was no relationship between fractionation period and HA yield. As such 2 h instead of the usual range of 12 to 24 h fractionation period could be considered optimum. Low ash content (5%), remarkable reduction in K, coupled with the fact that organic C, E4/E6, carboxylic COOH, phenolic OH, and total acidity values of the HA were consistent with those reported by other authors suggest that the HA dealt with were free from mineral matter. This was possible because the distilled water used to purify the HA served as Bronsted-Lowry acid during the purification process of the HA. Optimum purification period using distilled waster was 1 h instead of the usual range of 1 and 7 days (uses HF and HCl and dialysis). Humic acids could be isolated from tropical peat soils within 7 h (i.e. 4 h extraction, 2 h fractionation, and 1 h purification) instead of the existing period of 2 and 7 days. This could facilitate the idea of producing organic fertilizers such as ammonium-humate and potassium-humate from humified substances since techniques devised in this study did not alter the true nature of the HA. Besides, the technique is rapid, simple

  19. Urinary extracellular microvesicles: isolation methods and prospects for urinary proteome.

    Science.gov (United States)

    Wang, Danqi; Sun, Wei

    2014-08-01

    Extracellular microvesicles (EVs) are membranous vesicles, which are released from diverse cells. These EVs have also been found in a wide range of body fluids. The cargo of EVs, including proteins, lipids, carbohydrates, and nucleic acids, can be stably preserved in EVs. Researchers have found that EVs can mediate intercellular communication by shuttling the cargo components. Therefore, EVs can be used for the identification of disease-specific biomarkers. As one class of EVs, urinary exosomes can reflect the status of the renal system. Moreover, urinary exosome analysis can minimize the interference of high abundant proteins in the whole urine sample. Therefore, urinary exosomes have gained much attention in recent years. In this review, we present a comprehensive summary of urinary exosome studies in recent years, including collection, storage, and isolation methods. The normal and disease proteomic analyses of urinary exosomes are also presented. Thus, this review may provide a valuable reference for future research. PMID:24962155

  20. ISOL Targets Prepared with a New Paint Infiltration Coating Method

    CERN Document Server

    Kawai, Yoko; Kiggans, J O; Stracener, Dan

    2005-01-01

    A new infiltration paint coating method has been developed for fabricating ISOL targets for radioactive ion beam applications. The technique has been shown to be inexpensive, fast, and almost universal for the uniform deposition of many refractory target materials onto the interior surfaces of complex geometry matrices, such as Reticulated-Vitreous-Carbon-Foam (RVCF). The process yields robust, highly permeable targets with fast diffusion and release properties. We demonstrate the viability of the technique for coating forms of RVCF compressed by factors of 6 and 10 with materials to form targets for use at high energy facilities such as RIA. The use of compressed RVCF, coated with an optimum thickness of target material, reduces target lengths to practical values, while preserving high permeability. We calculate thermal conductivities and diffusion for various targets on 6xRVCF and 10xRVCF.

  1. Biosynthetic Pathway of the Reduced Polyketide Product Citreoviridin in Aspergillus terreus var. aureus Revealed by Heterologous Expression in Aspergillus nidulans.

    Science.gov (United States)

    Lin, Tzu-Shyang; Chiang, Yi-Ming; Wang, Clay C C

    2016-03-18

    Citreoviridin (1) belongs to a class of F1-ATPase β-subunit inhibitors that are synthesized by highly reducing polyketide synthases. These potent mycotoxins share an α-pyrone polyene structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution using heterologous expression in Aspergillus nidulans are reported. Two intermediates were isolated that allowed the proposal of the biosynthetic pathway of citreoviridin. PMID:26954888

  2. 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...

  3. Accessing natural product biosynthetic processes by mass spectrometry.

    Science.gov (United States)

    Bumpus, Stefanie B; Kelleher, Neil L

    2008-10-01

    Two important classes of natural products are made by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). With most biosynthetic intermediates covalently tethered during biogenesis, protein mass spectrometry (MS) has proven invaluable for their interrogation. New mass spectrometric assay formats (such as selective cofactor ejection and proteomics style LC-MS) are showcased here in the context of functional insights into new breeds of NRPS/PKS enzymes, including the first characterization of an 'iterative' PKS, the biosynthesis of the enediyne antitumor antibiotics, the study of a new strategy for PKS initiation via a GNAT-like mechanism, and the analysis of branching strategies in the so-called 'AT-less' NRPS/PKS hybrid systems. The future of MS analysis of NRPS and PKS biosynthetic pathways lies in adoption and development of methods that continue bridging enzymology with proteomics as both fields continue their post-genomic acceleration. PMID:18706516

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

    International Nuclear Information System (INIS)

    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

  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. Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

    Science.gov (United States)

    Yamamoto, Tsuyoshi; Matsui, Hidenori; Yamaji, Kenzaburo; Takahashi, Tetsufumi; Øverby, Anders; Nakamura, Masahiko; Matsumoto, Atsuko; Nonaka, Kenichi; Sunazuka, Toshiaki; Ōmura, Satoshi; Nakano, Hirofumi

    2016-09-01

    We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 μM), DHA (100 μM), or siamycin I (2.5 μM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis. PMID:27346378

  7. Structural Analysis Extended with Active Fault Isolation - Methods and Algorithms

    DEFF Research Database (Denmark)

    Gelso, Esteban R.; Blanke, Mogens

    2009-01-01

    Isolability of faults is a key issue in fault diagnosis whether the aim is maintenance or active fault-tolerant control. It is often encountered that while faults are detectable, they are only group-wise isolable from a usual diagnostic point of view. However, active injection of test signals on...... system inputs can considerably enhance fault isolability. This paper investigates this possibility of active fault isolation from a structural point of view. While such extension of the structural analysis approach was suggested earlier, algorithms and case studies were needed to explore this theory. The...

  8. 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...... bacteria and mine the associated chemical diversity. Thirteen strains closely related to Pseudoalteromonas luteoviolacea isolated from all over the Earth were analyzed using an untargeted metabolomics strategy, and metabolomic profiles were correlated with whole-genome sequences of the strains. We found......, integrative strategy for elucidating the chemical richness of a given set of bacteria and link the chemistry to biosynthetic genes....

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

    International Nuclear Information System (INIS)

    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)

  10. Comparison of rapid colorimetric method with conventional method in the isolation of mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Oberoi A

    2004-01-01

    Full Text Available The aim of the study was to evaluate two methods (colorimetric and conventional for isolation of Mycobacterium tuberculosis. A total of 500 clinical specimens were processed by modified Petroff′s method and then inoculated into MB/BacT-240 system bottles and on LJ medium slopes. The specimens included 242 sputum, 95 gastric aspirates, 47 pleural fluids, 45 CSF, 32 urine, 18 pus, 11 bronchoalveolar lavage, 3 tissue, 2 stool, 2 lymphnode specimens, 2 synovial fluid and 1 bronchial wash specimens. The isolation rate was 16.4% by the colorimetric method and 2.2% by the conventional method. The mean detection time was 16 days and 26 days respectively. Among 36 direct smear positive samples, 63.9%(23/36 and 30%(11/36 were positive by colorimetric and conventional methods respectively. Out of 464 direct smear negative samples 12.9%(60/464 and 0.6%(3/464 were positive by colorimetric and conventional methods respectively. Therefore, colorimetric method enables rapid detection leading to early diagnosis and drug susceptibility testing.

  11. Methods and Devices for Micro-Isolation, Extraction, and/or Analysis of Microscale Components

    Science.gov (United States)

    Kartalov, Emil P. (Inventor); Shibata, Darryl (Inventor); Taylor, Clive (Inventor); Wade, Lawrence A. (Inventor)

    2014-01-01

    Provided herein are devices and methods for the micro-isolation of biological cellular material. A micro-isolation apparatus described can comprise a photomask that protects regions of interest against DNA-destroying illumination. The micro-isolation apparatus can further comprise photosensitive material defining access wells following illumination and subsequent developing of the photosensitive material. The micro-isolation apparatus can further comprise a chambered microfluidic device comprising channels providing access to wells defined in photosensitive material. The micro-isolation apparatus can comprise a chambered microfluidic device without access wells defined in photosensitive material where valves control the flow of gases or liquids through the channels of the microfluidic device. Also included are methods for selectively isolating cellular material using the apparatuses described herein, as are methods for biochemical analysis of individual regions of interest of cellular material using the devices described herein. Further included are methods of making masking arrays useful for the methods described herein.

  12. Improved method for simultaneous isolation of proteins and nucleic acids.

    Science.gov (United States)

    Chey, Soroth; Claus, Claudia; Liebert, Uwe Gerd

    2011-04-01

    Guanidinium thiocyanate-phenol-chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol-bromochloropropane-water was used for precipitation of proteins from the phenol-ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample. PMID:21094121

  13. 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.

  14. Isolation, Structure Elucidation, Biosynthesis, and Synthesis of Antalid, a Secondary Metabolite from Polyangium species.

    Science.gov (United States)

    Tautz, Thomas; Hoffmann, Judith; Hoffmann, Thomas; Steinmetz, Heinrich; Washausen, Peter; Kunze, Brigitte; Huch, Volker; Kitsche, Andreas; Reichenbach, Hans; Höfle, Gerhard; Müller, Rolf; Kalesse, Markus

    2016-06-01

    The isolation, structure elucidation, and synthesis of antalid (1), a novel secondary metabolite from Polyangium sp., is described herein. The structure elucidation of 1 was performed with the aid of mass spectrometry, high field NMR experiments, and crystal structure analysis. The absolute configuration of antalid was confirmed through the Mosher ester method and ultimately by total synthesis. In addition, the biosynthetic origin of this hybrid PKS-NRPS natural product was unraveled by the in silico analysis of its biosynthetic gene cluster. PMID:27220069

  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)

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

    International Nuclear Information System (INIS)

    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

  17. 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.

  18. 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...

  19. Methods Comparison for Microsatellite Marker Development:Different Isolation Methods, Different Yield Efficiency

    Institute of Scientific and Technical Information of China (English)

    ZHAN Aibin; BAO Zhenmin; HU Xiaoli; LU Wei; HU Jingjie

    2009-01-01

    Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology, quantitative genetics and genomics. Therefore, it is extremely necessary to select several versatile, low-cost, efficient and time- and labor-saving methods to develop a large panel of microsatellite markers. In this study, we used Zhikong scallop (Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency, while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time- and cost-saving, it is difficult to obtain a large number of microsatellite markers, mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study, we recommend two methods, microsatellite-enriched library construction method and FIASCO-colony hybridization method, for large-scale microsatellite marker development. Both methods were derived from the mi-crosatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.

  20. Assessment of transport and isolation methods for gonococci.

    OpenAIRE

    Taylor, E.; Phillips, I

    1980-01-01

    Urethral discharge from men was diluted to give heavy and light inocula and cultured on seven different solid culture media, including two transport/isolation media, or held in three types of semi-solid transport medium for varying periods and then cultured. The amount of growth was quantitated and the performance of the different systems compared. Fresh non-selective media were best, with up to two failures in 254 cultures on each medium. With selective media there were 9-23 failures with he...

  1. Anthocyanin biosynthetic genes in Brassica rapa

    OpenAIRE

    Guo, Ning; Cheng, Feng; Wu, Jian; Liu, Bo; Zheng, Shuning; Liang, Jianli; Wang, Xiaowu

    2014-01-01

    Background Anthocyanins are a group of flavonoid compounds. As a group of important secondary metabolites, they perform several key biological functions in plants. Anthocyanins also play beneficial health roles as potentially protective factors against cancer and heart disease. To elucidate the anthocyanin biosynthetic pathway in Brassica rapa, we conducted comparative genomic analyses between Arabidopsis thaliana and B. rapa on a genome-wide level. Results In total, we identified 73 genes in...

  2. An eight-step synthesis of epicolactone reveals its biosynthetic origin

    Science.gov (United States)

    Ellerbrock, Pascal; Armanino, Nicolas; Ilg, Marina K.; Webster, Robert; Trauner, Dirk

    2015-11-01

    Epicolactone is a recently isolated fungal metabolite that is highly complex for its size, and yet racemic. With its array of quaternary stereocentres, high degree of functionalization and intricate polycyclic structure, it poses a considerable challenge to synthesis, a challenge that can be met by understanding its biosynthetic origin. If drawn in a certain way, epicolactone reveals a pattern that resembles purpurogallin, the archetype of ubiquitous natural colourants formed via oxidative dimerization. Based on this insight, we designed a biomimetic synthesis of epicolactone that proceeds in only eight steps from vanillyl alcohol. We have isolated a key intermediate that supports our biosynthetic hypothesis and anticipate that an isomer of epicolactone stemming from our synthetic efforts could also be found as a natural product.

  3. THE ISOLATION OF IONOGENIC SURFACTANTS FROM AQUEOUS SOIUTIONS BY PRECIPITATING AND SORPTION MICROFLOTATION METHODS

    OpenAIRE

    Streltsova, E. A.; Chromysheva, E. A.

    2016-01-01

    The manuscript is devoted to the establishment of colloid-chemical regularities of the ionogenic surfactants and flotation isolation process by precipitating and sorption microflotation methods in comparison with foam fractionating method and to the working out of the effective methods of their isolation from aqueous and technogenical solutions and sewage. The possibility of the surfactants (chlorides alkylammonium and alkylpyridinium, GIPH-3a, cetazole, pinazolyne, dodecylsulfate, sulfonole ...

  4. Arabidopsis cytochrome P450 cyp83B1 mutations activate the tryptophan biosynthetic pathway.

    OpenAIRE

    Smolen, Gromoslaw; Bender, Judith

    2002-01-01

    In plants, the tryptophan biosynthetic pathway provides a number of important secondary metabolites including the growth regulator indole-3-acetic acid (IAA) and indole glucosinolate defense compounds. Genes encoding tryptophan pathway enzymes are transcriptionally induced by a variety of stress signals, presumably to increase the production of both tryptophan and secondary metabolites during defense responses. To understand the mechanism of transcriptional induction, we isolated altered tryp...

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

    OpenAIRE

    Claudiu Iavornic; Gilbert-Rainer Gillich; Vasile Iancu; Zeno-Iosif Praisach; Ovidiu Vasile

    2011-01-01

    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.

  6. 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.

  7. High Quality DNA Isolation Method for Chickpea Genotypes

    OpenAIRE

    CİNGİLLİ, Hasibe; Abdülkadir AKÇİN

    2005-01-01

    In chickpea breeding genetic studies of individual plants need to be evaluated at the DNA level using molecular markers. A simple and reliable DNA extraction method is a prerequisite. This small-scale method is cetyltrimethylammonium bromide (CTAB)-based and extracts DNA from 1 to 3 folded young leaves processed in a 1.5 ml tube with 0.5 ml of extraction buffer and homogenized using an electric drill. Compared with the micro-prep method the improved mini-prep CTAB method is highly efficient a...

  8. Principal methods for isolation and identification of soil microbial communities.

    Science.gov (United States)

    Stefanis, Christos; Alexopoulos, Athanasios; Voidarou, Chrissa; Vavias, Stavros; Bezirtzoglou, Eugenia

    2013-01-01

    Soil microbial populations play crucial role in soil properties and influence below-ground ecosystem processes. Microbial composition and functioning changes the soil quality through decomposition of organic matter, recycling of nutrients, and biological control of parasites of plants. Moreover, the discovery that soil microbes may translate into benefits for biotechnology, management of agricultural, forest, and natural ecosystems, biodegradation of pollutants, and waste treatment systems maximized the need of scientists for the isolation and their characterization. Operations such as the production of antibiotics and enzymic activities from microorganisms of soil constitute objectives of industry in her effort to cope with the increase of population of earth and disturbance of environment and may ameliorate the effects of global climate change. In the past decades, new biochemical and molecular techniques have been developed in our effort to identify and classify soil bacteria. The goal of measuring the soil microbial diversity is difficult because of the limited knowledge about bacteria species and classification through families and orders. Molecular techniques extend our knowledge about microbial diversity and help the taxonomy of species. Measuring and monitoring soil microbial communities can lead us to better understanding of their composition and function in many ecosystem processes. PMID:22791233

  9. Comparison of methods of molybdenum isolation from spent catalyst

    International Nuclear Information System (INIS)

    Alternative methods are proposed of molybdenum precipitation from hydrochloric acid solutions (obtained during acidification of spent catalyst) as precipitations with higher amines or as MoS3 with degree of separation (from aqueous layer) 83-90 and 96-99 mass % respectively. For sulfuric acid media method of MoS3 precipitation with degree of separation 97-99 % is only suitable for variants considered. For molybdenum general separation from tarry organic residue its repeated washout by weak hydrochloric acid or sulfuric acid solutions is proposed

  10. Method for encapsulating and isolating hazardous cations, medium for encapsulating and isolating hazardous cations

    Energy Technology Data Exchange (ETDEWEB)

    Wasserman, S.R.; Anderson, K.B.; Song, K.; Yuchs, S.E.; Marshall, C.L.

    1996-12-31

    The problems associated with the disposal of toxic metals in an environmentally acceptable manner continues to plague industry. Such metals as nickel, vanadium, molybdenum, cobalt, iron, and antimony present physiological and ecological challenges that are best addressed through minimization of exposure and dispersion. A method for encapsulating hazardous cations is provided comprising supplying a pretreated substrate containing the cations; contacting the substrate with an organo-silane compound to form a coating on the substrate; and allowing the coating to cure. A medium for containing hazardous cations is also provided, comprising a substrate having ion-exchange capacity and a silane-containing coating on the substrate.

  11. Design-based re-engineering of biosynthetic gene clusters : plug-and-play in practice

    NARCIS (Netherlands)

    Frasch, Hans-Jörg; Medema, Marnix H.; Takano, Eriko; Breitling, Rainer; Gago, Federico; Parayil, Ajikumar

    2013-01-01

    Synthetic biology is revolutionizing the way in which the biosphere is explored for natural products. Through computational genome mining, thousands of biosynthetic gene clusters are being identified in microbial genomes, which constitute a rich source of potential novel pharmaceuticals. New methods

  12. Comprehensive curation and analysis of fungal biosynthetic gene clusters of published natural products.

    Science.gov (United States)

    Li, Yong Fuga; Tsai, Kathleen J S; Harvey, Colin J B; Li, James Jian; Ary, Beatrice E; Berlew, Erin E; Boehman, Brenna L; Findley, David M; Friant, Alexandra G; Gardner, Christopher A; Gould, Michael P; Ha, Jae H; Lilley, Brenna K; McKinstry, Emily L; Nawal, Saadia; Parry, Robert C; Rothchild, Kristina W; Silbert, Samantha D; Tentilucci, Michael D; Thurston, Alana M; Wai, Rebecca B; Yoon, Yongjin; Aiyar, Raeka S; Medema, Marnix H; Hillenmeyer, Maureen E; Charkoudian, Louise K

    2016-04-01

    Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, "activity-guided" approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent "genome mining" approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias toward the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and

  13. Molecular Diversity of Candida albicans Isolated from Immunocompromised Patients, Based on MLST Method

    Science.gov (United States)

    AFSARIAN, Seyed Mohammad Hosein; BADALI, Hamid; SHOKOHI, Tahereh; NAJAFIPOUR, Sohrab

    2015-01-01

    Background: As regards multilocus sequence typing (MLST) method directly analyze the polymorphism within DNA sequences; we performed the first nationwide study on the genotypic relationships of Candida albicans strains obtained from oropharynx and bronchoalveolar lavage (BAL) samples from immunocompromised patients. Methods: Fourteen epidemiologically unrelated clinical strains of C. albicans were obtained from three hospitals in Mazandaran Province, Iran (2006 to 2012) from seven patients with pulmonary infections and the rest with oropharyngeal samples of immunocompromised patients. Seven loci of housekeeping genes were sequenced for all fourteen isolates. Results: MLST was applied to a subset of 14 unrelated isolates. Seventy-one (2.5%) nucleotide sites were found to be variable. Accordingly, 60 different alleles were identified in seven loci among the isolates, among which two new alleles were obtained. Furthermore, 12 independent diploid sequence types (DSTs) including five novel DSTs were identified. The fourteen unrelated isolates were placed in 10 clonal clusters (CC) while two isolates were singletons, by eBURST analysis. Most of the isolates belonged to CC461 of eBURST analysis from the clade 11 and two isolates assigned to CC172 from the clade 15. Conclusion: Pathogen distribution and relatedness for determining the epidemiology of nosocomial infections is highly recommended for pathogen control methods. PMID:26587501

  14. Rapid Pulsed-Field Gel Electrophoresis Method for Group B Streptococcus Isolates

    OpenAIRE

    Benson, Jeffrey A.; Ferrieri, Patricia

    2001-01-01

    We developed a rapid pulsed-field gel electrophoresis (PFGE) method that required 3 days to complete, an improvement over the standard method that required as many as 8 days. The accuracy and reproducibility of the rapid method were verified by analysis of DNA band sizes of our control group B streptococcus isolate. The rapid method was superior to the standard method, providing more precise molecular sizing and gels of higher image quality. The reproducibility of rapid PFGE substantiated its...

  15. Investigation of the biosynthetic potential of endophytes in traditional Chinese anticancer herbs.

    Directory of Open Access Journals (Sweden)

    Kristin I Miller

    Full Text Available Traditional Chinese medicine encompasses a rich empirical knowledge of the use of plants for the treatment of disease. In addition, the microorganisms associated with medicinal plants are also of interest as the producers of the compounds responsible for the observed plant bioactivity. The present study has pioneered the use of genetic screening to assess the potential of endophytes to synthesize bioactive compounds, as indicated by the presence of non-ribosomal peptide synthetase (NRPS and polyketide synthase (PKS genes. The total DNA extracts of 30 traditional Chinese herbs, were screened for functional genes involved in the biosynthesis of bioactive compounds. The four PCR screens were successful in targeting four bacterial PKS, six bacterial NRPS, ten fungal PKS and three fungal NRPS gene fragments. Analysis of the detected endophyte gene fragments afforded consideration of the possible bioactivity of the natural products produced by endophytes in medicinal herbs. This investigation describes a rapid method for the initial screening of medicinal herbs and has highlighted a subset of those plants that host endophytes with biosynthetic potential. These selected plants can be the focus of more comprehensive endophyte isolation and natural product studies.

  16. EQUIVALENT EXCITATION METHOD FOR VIBRATION ISOLATION DESIGN:THEORETICAL ANALYSIS AND EXPERIMENTAL RESULTS

    Institute of Scientific and Technical Information of China (English)

    Huo Rui; Shi Yin

    2005-01-01

    In view of difficulties concerned with direct measurement of excitations inside source equipments and their significant influence on vibration isolation effectiveness, a dynamical model, for vibration isolation of a rigid machine with six-degree-of-freedom mounted on a flexible foundation through multiple mounts, is analyzed, in which the complicated and multiple disturbances inside the machine are described as an equivalent excitation spectrum. And a method for the estimation of the equivalent excitation spectrum according to system dynamic responses is discussed for the quantitative prediction of isolation effectiveness.Both theoretical analysis and experimental results are demonstrated. Further work shows the quantitative prediction of transmitted power flow in a flexible vibration isolation experiment system using the proposed equivalent excitation spectrum method, by comparison with its testing results.

  17. Comparison of isolation and quantification methods to measure humic-like substances (HULIS) in atmospheric particles

    Science.gov (United States)

    Fan, Xingjun; Song, Jianzhong; Peng, Ping'an

    2012-12-01

    Humic-like Substances (HULIS) comprise a significant fraction of the water-soluble organic aerosol mass and influence the cloud microphysical properties and climate effects of aerosols in the atmosphere. In this work, the most frequently used HULIS isolation and quantification methods including ENVI-18, HLB, XAD-8 and DEAE were comparatively characterized with two model standards, ten interfering compounds, and five ambient aerosol samples. Quantification of HULIS is performed with a TOC analyzer, complemented by an investigation of the chemical structure of the extracted fractions by UV-Vis spectroscopy. The results show that the four isolation methods were all characterized by high reliability, high reproducibility, and low limit of detection (LOD), indicating that each method can be used to efficiently recover Suwannee River Fulvic Acid (SRFA) and be applied to the quantification of the lower amount of HULIS in atmospheric particles. The analytical results of the UV-Vis spectra of HULIS fractions isolated also indicate that they are all favorable for extraction of compounds of high UV absorbance, high MW, and high aromaticity and that the DEAE protocol is the most significant one. Compared with the DEAE method that favors extraction of highly UV-absorbing and more aromatic compounds, SRFA isolated by the ENVI-18, HLB, and XAD-8 protocols were more representative of the global matrix. Each method has its own advantages and disadvantages and is suitable for a particular application. No single method is ideal for both isolation and quantification of HULIS in atmospheric samples.

  18. Biosynthetic Pathway for the Epipolythiodioxopiperazine Acetylaranotin in Aspergillus terreus Revealed by Genome-based Deletion Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Chun-Jun; Yeh, Hsu-Hua; Chiang, Yi Ming; Sanchez, James F.; Chang, ShuLin; Bruno, Kenneth S.; Wang, Clay C.

    2013-04-15

    Abstract Epipolythiodioxopiperazines (ETPs) are a class of fungal secondary metabolites derived from cyclic peptides. Acetylaranotin belongs to one structural subgroup of ETPs characterized by the presence of a seven-membered dihydrooxepine ring. Defining the genes involved in acetylaranotin biosynthesis should provide a means to increase production of these compounds and facilitate the engineering of second-generation molecules. The filamentous fungus Aspergillus terreus produces acetylaranotin and related natural products. Using targeted gene deletions, we have identified a cluster of 9 genes including one nonribosomal peptide synthase gene, ataP, that is required for acetylaranotin biosynthesis. Chemical analysis of the wild type and mutant strains enabled us to isolate seventeen natural products that are either intermediates in the normal biosynthetic pathway or shunt products that are produced when the pathway is interrupted through mutation. Nine of the compounds identified in this study are novel natural products. Our data allow us to propose a complete biosynthetic pathway for acetylaranotin and related natural products.

  19. Improved method for isolation of coupled mitochondria of Araucaria angustifolia (Bert.) O. Kuntze

    OpenAIRE

    André Bellin Mariano; Leonardo Kovalhuk; Caroline Valente; Juliana Maurer-Menestrina; Adaucto Bellarmino de Pereira-Netto; Miguel Pedro Guerra; Eva Gunilla Skare Carnieri

    2004-01-01

    A method for the isolation of coupled mitochondria from the callus of Araucaria angustifolia is described for the first time. Mitochondria were isolated from embryogenic callus of A. angustifolia. They were metabolically active, able to sustain oxidative phosphorylation as shown by respiratory control ratio values, which were about 2.4 when respiring on succinate as substrate. Oxygen uptake experiments, using freeze-thawed disrupted mitochondria, showed the presence of alternative rotenone-in...

  20. A rapid and simple method for the isolation of pure eosinophilic leukocytes from horse blood.

    Science.gov (United States)

    Jörg, A; Portmann, P; Fellay, G; Dreyer, J L; Meyer, J

    1978-12-15

    An improved and short method is described for the isolation of intact eosinophilic leukocytes from horse blood with high yield (1--1.5 g/20 l). Viability and purity of the preparations were verified by light and electron microscopy and by the trypan blue exclusion test. Isolated eosinophils were 98--100% pure, intact and viable, and they could be shown to phagocytise immune-complexes. PMID:729750

  1. Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Engberg, J.; Fussing, V.;

    2000-01-01

    Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (Ribo...

  2. Ion exchange method for calcium isolation from solutions of zinc, cadmium, and cobalt salts

    International Nuclear Information System (INIS)

    A method for isolating impurity amounts of calcium from zinc, cadmium and cobalt salts has been suggested. The method consists in dissolution of the above-mentioned metal salts in ammonia solution, selective sorption of calcium impurities from solutions prepared on carboxylic cationite with subsequent calcium desorption by mineral acid solution. 4 refs.; 2 figs

  3. 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

  4. Evaluation of different detection methods of biofilm formation in the clinical isolates

    Directory of Open Access Journals (Sweden)

    Afreenish Hassan

    2011-08-01

    Full Text Available BACKGROUND: Microorganisms growing in a biofilm are associated with chronic and recurrent human infections and are highly resistant to antimicrobial agents. There are various methods to detect biofilm production like Tissue Culture Plate (TCP, Tube method (TM, Congo Red Agar method (CRA, bioluminescent assay, piezoelectric sensors, and fluorescent microscopic examination. OBJECTIVE: This study was conducted to compare three methods for the detection of biofilms. METHOD: The study was carried out at the Department of Microbiology, Army Medical College, National University of Sciences and Technology, Pakistan, from January 2010 to June 2010. A total of 110 clinical isolates were subjected to biofilm detection methods. Isolates were identified by standard microbiological procedures. Biofilm detection was tested by TCP, TM and CRA. Antibiotic susceptibility test of biofilm producing bacteria was performed by using the Kirby-Bauer disc diffusion technique according to CLSI guidelines. RESULTS: The TCP method was considered to be superior to TM and CRA. From the total of 110 clinical isolates, TCP method detected 22.7% as high, 41% moderate and 36.3% as weak or non-biofilm producers. We have observed higher antibiotic resistance in biofilm producing bacteria than non-biofilm producers. CONCLUSION: We can conclude from our study that the TCP method is a more quantitative and reliable method for the detection of biofilm forming microorganisms as compared to TM and CRA methods, and it can be recommended as a general screening method for detection of biofilm producing bacteria in laboratories.

  5. Improved explant method to isolate umbilical cord-derived mesenchymal stem cells and their immunosuppressive properties.

    Science.gov (United States)

    Mori, Yuka; Ohshimo, Jun; Shimazu, Takahisa; He, Haiping; Takahashi, Atsuko; Yamamoto, Yuki; Tsunoda, Hajime; Tojo, Arinobu; Nagamura-Inoue, Tokiko

    2015-04-01

    The umbilical cord (UC) has become one of the major sources of mesenchymal stem cells (MSCs). The common explant method of isolating UC-derived MSCs (UC-MSCs) involves mincing the UCs into small fragments, which are then attached to a culture dish bottom from which the MSCs migrate. However, the fragments frequently float up from the bottom of the dish, thereby reducing the cell recovery rate. To overcome this problem, we demonstrate an improved explant method for UC-MSC isolation, which involves the use of a stainless steel mesh (Cellamigo(®); Tsubakimoto Chain Co.), to protect the tissue from floating after the minced fragments are aligned at regular intervals in culture dishes. The culture medium was refreshed every 3 days and the adherent cells and tissue fragments were harvested using trypsin. The number of UC-MSCs isolated from 1 g of UC using the explant method with Cellamigo was 2.9 ± 1.4 × 10(6)/g, which was significantly higher than that obtained without Cellamigo (0.66 ± 0.53 × 10(6)/g) (n = 6, p < 0.01) when cells reached 80-90% confluence. In addition, the processing and incubation time required to reach 80-90% confluence was reduced in the improved explant method compared with the conventional method. The UC-MSCs isolated using the improved method were positive for CD105, CD73, CD90, and HLA class I expression and negative for CD45 and HLA class II expression. The isolated UC-MSCs efficiently inhibited the responder T cells induced by allogeneic dendritic cells in a mixed lymphocyte reaction. Conclusively, we demonstrated that the use of Cellamigo improves the explant method for isolating UC-MSCs. PMID:25220032

  6. Influence of DNA isolation method on the investigation of archaeal diversity and abundance in biogas plants.

    Science.gov (United States)

    Theiss, Juliane; Rother, Michael; Röske, Kerstin

    2016-09-01

    Various methods are available for DNA isolation from environmental samples. Because the chemical and biological composition of samples such as soil, sludge, or plant material is different, the effectiveness of DNA isolation can vary depending on the method applied and thus, have a substantial effect on the results of downstream analysis of the microbial community. Although the process of biogas formation is being intensely investigated, a systematic evaluation of kits for DNA isolation from material of biogas plants is still lacking. Since no DNA isolation kit specifically tailored for DNA isolation from sludge of biogas plants is available, this study compares five commercially available kits regarding their influence on downstream analyses such denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR). The results show that not all kits are equally suited for the DNA isolation from samples of different biogas plants, but highly reproducible DGGE fingerprints as well as qPCR results across the tested samples from biogas reactors using different substrate compositions could be produced using selected kits. PMID:27089887

  7. Development of an evaluation method for seismic isolation systems of nuclear power facilities. Probabilistic failure assessment method of crossover piping

    International Nuclear Information System (INIS)

    In this paper, the probabilistic failure assessment method for crossover piping is addressed. The crossover piping in this study having universal flexible joint for low temperature and low pressure piping system, is installed between seismically isolated and non-isolated building; the reactor building is only isolated using seismic isolation systems whereas the turbine building is non-isolated. The crossover piping is designed to meet the large displacement for seismic event. It is known that the major effect on such crossover piping by seismic event is fatigue damage. The failure probability by fatigue is evaluated as followings. The equivalent numbers of cycles for maximum stress (SR) on universal flexible joint is calculated by Rainflow-counting method. The SRs are distributed on two fatigue curves as 'Design fatigue curve' by JSME and 'Best-Fit fatigue curve'. When the distributions are on the former curve can be read as 99% confidence with enough design margins and the distributions are on the latter curve can be read as 50% probabilistic failure of bearing force. In conclusion, those failure probabilities distributions can be fit to fragility curve. (author)

  8. Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates.

    Science.gov (United States)

    Nagy, Elisabeth; Justesen, Ulrik Stenz; Eitel, Zsuzsa; Urbán, Edit

    2015-02-01

    With the emergence of antibiotic resistance among Bacteroides fragilis group isolates the need of susceptibility testing in routine laboratories is increasing. The aims of the present study were to evaluate the disk diffusion method for susceptibility testing in case of different clinical isolates of Bacteroides spp by comparing zone diameter results with MICs obtained earlier during an Europe-wide antibiotic susceptibility surveillance, and to propose zone diameter breakpoints, which correlate for the EUCAST MIC breakpoints. We tested 381 clinical isolates of the B. fragilis group to amoxicillin/clavulanic acid, cefoxitin, clindamycin, imipenem, metronidazole, moxifloxacin, piperacillin/tazobactam, tigecycline by agar dilution method previously. The inhibition zones of the same antibiotics including meropenem disc were determined by the disc diffusion on Brucella blood agar supplemented with haemin and vitamin K1. Plates were incubated at 37 °C in an anaerobic atmosphere for 24 h. The zone diameters were read at 100% inhibition. In case of discrepant results MICs were determined by gradient test and compared with the inhibition zones on the same plate. We found a good agreement between the inhibition zone diameters and the MICs for imipenem, metronidazole, moxifloxacin and tigecyclin. The inhibition zone diameters of meropenem also separated clearly the isolates, which can be considered wild-type isolates. In case of amoxicillin/clavulanic acid and piperacillin/tazobactam intermediate and susceptible isolates according to the MIC determination, overlap during the zone diameter determination. Isolates with an inhibition zone disk diffusion can be an option for susceptibility testing of B. fragilis group isolates for most relevant antibiotics in routine laboratories. PMID:25464140

  9. Detection of vancomycin susceptibility among clinical isolates of MRSA by using minimum inhibitory concentration method

    Directory of Open Access Journals (Sweden)

    P. Sreenivasulu Reddy

    2015-06-01

    Full Text Available Background: Staphylococcus aureus is considered as a major pathogen causing a diversity of infections including bacteremia, pneumonia, skin and soft tissue including osteoarticular infections. Since 1961, Methicillin Resistant Staphylococci aureus (MRSA emerged has one of the major and common cause of hospital acquired infection. However, due to wide spread usage of vancomycin for MRSA infections resulted in reduced susceptibility of S. aureus to vancomycin has been identified as a serious public health concern. The aim of the study is to identify the Methicillin Resistant Staphylococcus aureus (MRSA from various clinical samples and to detect vancomycin susceptibility by Minimum Inhibitory Concentration (MIC method. Methods: This study was conducted over period of one year December 2013 to November 2014. Clinical samples like pus, blood, sputum, urine and cerebrospinal fluid were collected from various clinical departments in Narayana General Hospital for selective isolation of Staphylococcus aureus. A total of 100 Staphylococcal aureus isolates were isolatedby using standard laboratory procedures. MRSA were detected using Oxacillin Disc on Muller Hinton Agar with 4% NaCl. Sensitivity pattern for vancomycin (30 and micro;g disc and for other recommended antibiotics was determined by Kirby-Bauer's disk diffusion method. Minimum Inhibitory Concentration (MIC was done for vancomycin sensitive isolates by standard agar dilution method. Results: Out of 100 S. aureus isolates, all were susceptible to vancomycin (30 and micro;g by disk diffusion method. But, 82 isolates of MRSA were susceptible to vancomycin at the concentration of 0.5-2 and #956;g/ml of agar. 17 isolates showed intermediate sensitivity to vancomycin, in which 13 isolates with MIC 4 and #956;g/ml and 4 isolates with MIC 8 and #956;g/ml and one isolate was resistant to vancomycin even with MIC of 16 and #956;g/ml. Conclusions: The present study reveals the emergence of Vancomycin

  10. 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.

  11. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    Science.gov (United States)

    Tolios, Alexander; Teupser, Daniel; Holdt, Lesca M

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements. PMID:26636575

  12. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    Directory of Open Access Journals (Sweden)

    Alexander Tolios

    Full Text Available Telomeres are located at chromosome ends and their length (TL has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen, spin columns (Qiagen, Macherey-Nagel and 5prime or precipitation (Stratec/Invisorb and a published isopropanol precipitation protocol (IPP were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen. Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime. Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.

  13. Highly efficient method for isolation of total RNA from adipose tissue

    DEFF Research Database (Denmark)

    Cirera Salicio, Susanna

    2013-01-01

    RNeasy method results in cleaner samples but more prone to degradation while the TRI Reagent(R) method results in samples contaminated with salts and solvents but more intact. The new protocol combines the best of both methods resulting in RNA of high quality and suitable for downstream experiments like RT......-qPCR, microarrays and high-throughput sequencing. Conclusions: The current protocol for total RNA isolation from adipose tissue yields sufficient amount of high quality total RNA free of contaminants....

  14. Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

    Science.gov (United States)

    Taylor, Douglas D; Shah, Sahil

    2015-10-01

    Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and

  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. PMID:27005523

  16. 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.

  17. A modified Phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients

    Directory of Open Access Journals (Sweden)

    Clemens Hufnagl

    2013-03-01

    Full Text Available Searching for new cancer biomarkers, circulating cell-free DNA (cfDNA has become an appealing target of interest as an elevated level of cfDNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since cfDNA can be isolated from the circulation and other body fluids of patients without harming their physical condition, cfDNA is becoming a promising candidate as a novel non-invasive biomarker for cancer. The challenge in the diagnostic analysis of cfDNA is its very low presence in human plasma/serum and its partially strong fragmentation. Here we evaluated a modified phenol/chloroform extraction method for the isolation of cfDNA and compared it with published standard methods for cfDNA isolation.

  18. A method for rapid isolation of total RNA of high purity and yield from Arthrospira platensis.

    Science.gov (United States)

    Pathak, Ravi Ramesh; Lochab, Sunila

    2010-07-01

    Arthrospira (Spirulina) platensis is widely used as a food supplement and has been an economically important species for centuries. However, the genetic aspect of studies of this particular organism has always been neglected, mainly because of the nonavailability of suitable methods for isolation of nucleic acids and the difficulties faced during further manipulations. Although total RNA has been isolated using commercially available kits, we present a method optimized to obtain DNA-free total RNA of higher yields and higher purity in less time than is required by other methods (precipitation and 70% ethanol wash. This method, optimized for the cyanobacterium Arthrospira (Spirulina) platensis, eliminates the need for DNase treatment and produces high-quality RNA, as validated by bioanalyzer, RT-PCR, and cloning. With the recent release of the Arthrospira genome, the current method will be of great value for carrying out high-throughput studies like microarray and real-time PCR. PMID:20651857

  19. Isolation, characterization and optimization of indigenous acetic acid bacteria and evaluation of their preservation methods

    Directory of Open Access Journals (Sweden)

    K Beheshti-Maal

    2010-06-01

    Full Text Available Background and Objectives: Acetic acid bacteria (AAB are useful in industrial production of vinegar. The present study aims at isolation and identification of acetic acid bacteria with characterization, optimization, and evaluation of their acetic acid productivity."nMaterials and Methods: Samples from various fruits were screened for presence of acetic acid bacteria on glucose, yeast extract, calcium carbonate (GYC medium. Carr medium supplemented with bromocresol green was used for distinguishing Acetobacter from Gluconobacter. The isolates were cultured in basal medium to find the highest acetic acid producer. Biochemical tests followed by 16S rRNA and restriction analyses were employed for identification of the isolate and phylogenic tree was constructed. Bacterial growth and acid production conditions were optimized based on optimal inoculum size, pH, temperature, agitation, aeration and medium composition."nResults: Thirty-seven acetic acid bacteria from acetobacter and gluconobacter members were isolated. Acetic acid productivity yielded 4 isolates that produced higher amounts of acid. The highest producer of acid (10.03% was selected for identification. The sequencing and restriction analyses of 16S rRNA revealed a divergent strain of Acetobacter pasteurianus (Gene bank accession number # GU059865. The optimum condition for acid production was a medium composed of 2% glucose, 2% yeast extract, 3% ethanol and 3% acid acetic at inoculum size of 4% at 3L/Min aeration level in the production medium. The isolate was best preserved in GYC medium at 12oC for more than a month. Longer preservation was possible at -70oC."nConclusion: The results are suggestive of isolation of an indigenous acetic acid bacteria. Pilot plan is suggested to study applicability of the isolated strain in acetic acid production.

  20. 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.

  1. Coumarins in horse chestnut flowers: isolation and quantification by UPLC method.

    Science.gov (United States)

    Dudek-Makuch, Marlena; Matławska, Irena

    2013-01-01

    The coumarins: scopoletin, esculetin and fraxetin were isolated from the flowers of horse chestnut (Aesculus hippocastanum L., Hippocastanaceae) and identified by spectrophotometric methods (UV, 1H, 13C NMR, ESI-MS). Their content, determined using the Ultra Performance Liquid Chromatography (UPLC), was 0.41, 0.13 and 0.05%, respectively. PMID:23757942

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

    Science.gov (United States)

    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...

  3. AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus

    Directory of Open Access Journals (Sweden)

    Sirintip Dangtip

    2015-11-01

    Full Text Available Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VPAHPND revealed that it was mediated by a binary Pir-like toxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPND-causing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences of ToxA, a one-step PCR detection method for VPAHPND was introduced in June 2014 but had the limitation that attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VPAHPND in shrimp or other samples could not be detected without first preparing an enrichment broth culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4 method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VPAHPND using a panel of 104 bacterial isolates including 51 VPAHPND isolates and 53 non-AHPND isolates, the latter including 34 isolates of V. parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species. The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template than the one-step AP3 (10 pg total DNA template method, and it could detect VPAHPND in experimentally challenged shrimp by 6 h post immersion (n = 2/3, while AP3 could not detect is until 12 h post immersion (n = 1/3. Thus, the AP4 method may be useful in detecting VPAHPND isolates in samples where target material is limited (e.g., small tissue quantity or archived DNA and enrichment cannot be employed (i.e., frozen samples or samples preserved in alcohol.

  4. Comparison of epidemiological marker methods for identification of Salmonella typhimurium isolates from an outbreak caused by contaminated chocolate.

    OpenAIRE

    Kapperud, G; Lassen, J.; Dommarsnes, K; Kristiansen, B. E.; Caugant, D A; Ask, E; Jahkola, M

    1989-01-01

    Plasmid profile analysis, restriction endonuclease analysis, and multilocus enzyme electrophoresis were used in conjunction with serotyping, bacteriophage typing, and biochemical fingerprinting to trace epidemiologically related isolates of Salmonella typhimurium from an outbreak caused by contaminated chocolate products in Norway and Finland. To evaluate the efficiency of the epidemiological marker methods, isolates from the outbreak were compared with five groups of control isolates not kno...

  5. A method for the large scale isolation of high transformation efficiency fungal genomic DNA.

    Science.gov (United States)

    Zhang, D; Yang, Y; Castlebury, L A; Cerniglia, C E

    1996-12-01

    A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported. This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature. The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA. Sau3AI partially digested DNA showed high transformation efficiency (> 10(6)/100 ng DNA) when ligated to ZAP-express lambda vector. PMID:8961565

  6. Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus

    Science.gov (United States)

    Lasure, Linda L. [Fall City, WA; Dai, Ziyu [Richland, WA

    2008-10-21

    The invention includes isolated polynucleotide molecules that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention includes a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. The polynucleotide sequence is expressed to promote a first morphology. The first morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a second morphology.

  7. Isolation of Entomopathogenic Fungi From Soils and Ixodes scapularis (Acari: Ixodidae) Ticks: Prevalence and Methods

    OpenAIRE

    Tuininga, Amy R.; Miller, Jessica L.; Shannon U. Morath; Daniels, Thomas J.; Falco, Richard C.; Marchese, Michael; Sahabi, Sadia; Rosa, Dieshia; Stafford, Kirby C.

    2009-01-01

    Entomopathogenic fungi are commonly found in forested soils that provide tick habitat, and many species are pathogenic to Ixodes scapularis Say, the blacklegged tick. As a first step to developing effective biocontrol strategies, the objective of this study was to determine the best methods to isolate entomopathogenic fungal species from field-collected samples of soils and ticks from an Eastern deciduous forest where I. scapularis is common. Several methods were assessed: (1) soils, leaf lit...

  8. Simple Method for Detection of Metallo – β–Lactamase Among Gram Negative Isolates

    OpenAIRE

    Agrawal R; Sumana MN; Kishore A; Kulkarni M

    2015-01-01

    Background: Carbapenem resistance due to the production of metallo-β -lactamase (MBL) in Gram-negative organism is an increasing public health problem. Aim: The purpose of this study is to detectMBL in Gram Negative bacterial isolates among Ventilator Associated Pneumonia patients. Materials and Methods: Phenotypic detection of MBL was done by three methods: 1.Modified Hodge Test (MHT) 2.Combined disc test (CDT) 3.Double Disc Synergy Test (DDST). Results: Out of 126 gram negative bacterial is...

  9. Evaluation of antifungal susceptibility testing in Candida isolates by Candifast and disk-diffusion method

    OpenAIRE

    Sidhartha Giri; Anupma Jyoti Kindo

    2014-01-01

    With the increase in invasive fungal infections due to Candida species and resistance to antifungal therapy, in vitro antifungal susceptibility testing is becoming an important part of clinical microbiology laboratories. Along with broth microdilution and disk diffusion method, various commercial methods are being increasingly used for antifungal susceptibility testing, especially in the developed world. In our study, we compared the antifungal susceptibility patterns of 39 isolates of Candid...

  10. A method of isoflavones isolation from red clover as standards for analyses

    Directory of Open Access Journals (Sweden)

    Piotr M. Górski

    2013-12-01

    Full Text Available Five compounds having an isoflavone structure were isolated from the tops of red clover (Trifolium pratense. On the basis of spectral (UV, MS and chromatographic (TLC, HPLC analyses the compounds were identified as biochanin A, formononetin, pratensein, genistein and daidzein. Biochanin A and formononetin - two main clover estrogens - were obtained in crystalline forms in the amounts of 50 mg and 15 mg, respectively (per 250 g of D. W.. Homogenous fractions of pratensein, genistein, and daidzein were also obtained. The obtained standards were used as markers for peak identification in HPLC. The presented method of isoflavone isolation is simple and quick. It allows for the simple isolation of a mixture of crude isoflavone aglycones which may be used in qualitative and semi-quantitative (TLC analyses. The individual standards are obtained through column chromatography and may be used for quantitative analyses.

  11. An effective method of RNA isolation from Fallopia multiflora tuberous roots.

    Science.gov (United States)

    Chen, Lei; Sheng, Shu-Jing; Tan, Xue-Mei; Shen, Yan-Jing; Li, Hong-Qing; Zhao, Shu-Jin

    2012-01-01

    To isolate high-quality total RNA from Fallopia multiflora tuberous roots is difficult because of the presence of high levels of carbohydrates, phenolics, and other secondary metabolites. Since several procedures specialized for RNA isolation from polysaccharides and phenols rich tissues have resulted in poor yields, in this study, we developed a modified protocol that was derived from the traditional CTAB method. The protocol was able to produce high-quality and intact RNA from the tuberous roots of F. multiflora. The yield of total RNA was more than 0.15 mg/g fresh weight, with an A260/A280 ratio of 1.9-2.0. The obtained RNA was of sufficient quality and suitable for downstream application such as reverse-transcription polymerase chain reaction (RT-PCR), Northern hybridization, and cDNA library construction. The protocol may also have wider applicability for total RNA isolation from other plant species with tuberous roots. PMID:22239710

  12. Cyclic Lipopeptide Biosynthetic Genes and Products, and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria

    OpenAIRE

    Mora, Isabel; Cabrefiga, Jordi; Montesinos, Emilio

    2015-01-01

    The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (9...

  13. Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

    OpenAIRE

    Barghouthi, S; Payne, S M; Arceneaux, J E; Byers, B R

    1991-01-01

    Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete n...

  14. A simplified but robust method for the isolation of avian and mammalian muscle satellite cells

    Directory of Open Access Journals (Sweden)

    Baquero-Perez Belinda

    2012-06-01

    Full Text Available Abstract Background Current methods of isolation of muscle satellite cells from different animal species are highly variable making inter-species comparisons problematic. This variation mainly stems from the use of different proteolytic enzymes to release the satellite cells from the muscle tissue (sometimes a single enzyme is used but often a combination of enzymes is preferred and the different extracellular matrix proteins used to coat culture ware. In addition, isolation of satellite cells is frequently laborious and sometimes may require pre-plating of the cell preparation on uncoated flasks or Percoll centrifugation to remove contaminating fibroblasts. The methodology employed to isolate and culture satellite cells in vitro can critically determine the fusion of myoblasts into multi-nucleated myotubes. These terminally differentiated myotubes resemble mature myofibres in the muscle tissue in vivo, therefore optimal fusion is a keystone of in vitro muscle culture. Hence, a simple method of muscle satellite cell isolation and culture of different vertebrate species that can result in a high fusion rate is highly desirable. Results We demonstrate here a relatively simple and rapid method of isolating highly enriched muscle satellite cells from different avian and mammalian species. In brief, muscle tissue was mechanically dissociated, digested with a single enzyme (pronase, triturated with a 10-ml pipette, filtered and directly plated onto collagen coated flasks. Following this method and after optimization of the cell culture conditions, excellent fusion rates were achieved in the duck, chicken, horse and cow (with more than 50% cell fusion, and to a lesser extent pig, pointing to pronase as a highly suitable enzyme to release satellite cells from muscle tissue. Conclusions Our simplified method presents a quick and simple alternative to isolating highly enriched muscle satellite cell cultures which can subsequently rapidly differentiate

  15. 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)

    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)

  16. Application of retrograde dissection method for isolation of bone marrow cells from rat femurs and tibiae.

    Science.gov (United States)

    Li, C M; Fu, B M; Zhang, L C; Tang, B; Zhu, L; Zhao, Y; Zhang, J

    2016-01-01

    Currently, there is no practical and efficient method for the isolation of bone marrow cells (BMCs) from rat femurs and tibiae. Here, we attempted to develop a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae. Rat femurs and tibiae were dissected from the ankle to the hip joint; subsequently, a three-step "locate-slide-twist" procedure was performed using scissors and forceps to remove the femurs and tibiae completely, from the surrounding musculature. The bones were flushed with phosphate-buffered saline to harvest BMCs. The femurs and tibiae were dissected in 1.8 ± 0.6 min, and the BMC suspension preparation time was 13.1 ± 2.3 min. The bone marrow cavities did not incur any fractures or injuries during the isolation. Culture of harvested BMCs for 72 h led to a significant increase in cell number from 4.4 ± 0.3 x 106 to 6.9 ± 0.7 x 10(6) (P 0.05). Microscopic examination of the isolated BMCs after the 72-h incubation period revealed the no-microbial or muscle cell contamination. Furthermore, flow cytometry revealed that cultured BMCs (72-h culture) grew well. Here, we have reported a rapid, simple, effective, and non-contaminating method for the isolation of BMCs from rat femurs and tibiae by using retrograde dissection. This method can be used to harvest a large number of viable BMCs without the risk of contamination from muscle and connective tissues. PMID:27323101

  17. Molecular characterization of Staphylococcus aureus isolates from southwest of Iran using spa and SCCmec typing methods.

    Science.gov (United States)

    Darban-Sarokhalil, Davood; Khoramrooz, Seyed Sajjad; Marashifard, Masoud; Malek Hosseini, Seyed Ali Asghar; Parhizgari, Najmeh; Yazdanpanah, Mahboobeh; Gharibpour, Farzaneh; Mirzaii, Mehdi; Sharifi, Bahman; Haeili, Mehri

    2016-09-01

    Staphylococcus aureus remains a major cause of nosocomial infection worldwide. Characterization of S. aureus isolates circulating in the southwest of Iran will contribute to understand and control the spread of the strains in this area. spa and SCCmec typing methods were used for genotyping of 125 S. aureus isolates obtained from two teaching hospitals in Ahvaz. Drug susceptibility testing was performed by using disk diffusion method. Frequency of the methicillin resistant S. aureus (MRSA) isolates was 39% (n = 34) and 27% (n = 10) in Emam Khomeini and Golestan hospitals, respectively. Except for Erythromycin, MRSA strains showed high rate of resistance to antimicrobial agents including penicillin (100%), norfloxacine (80%), azitromycin (80%), ciprofloxacin (80%), gentamycin (77%), cotrimoxazole (75%), cephotaxime. All isolates were sensitive to vancomycin. Out of 44 MRSA strains, 39 (88.5%) were SCCmec III, three (7%) were IVc and two (4.5%) of them were nontypeable. spa types t037 (26 isolates; 59%), and t1149 (25 isolates; 31%) were the most dominant types found in MRSA and methicillin sensitive S. aureus (MSSA) strains, respectively. We found SCCmec type III as the most prominent type indicating that most of the studied bacterial population had hospital origin. spa type t037, the most frequent genotype in this study were significantly (100%) associated with MRSA. For the first time we are reporting spa types t692, t706 and t018 from Iran and t342, t704, t2622, t5598, t11270 and t2864 from Asia. Moreover we are reporting types t6871 and t2684 for the second time in the world. PMID:27392699

  18. A highly efficient miPCR method for isolating FSTs from transgenic Arabidopsis thaliana plants

    Indian Academy of Sciences (India)

    Gennady V. Pogorelko; Oksana V. Fursova

    2008-08-01

    The exact localization of an insertion in the genome of transgenic plants obtained by Agrobacterium-mediated transformation is an integral part of most experiments aimed at studying these types of mutants. There are several methods for isolating unknown nucleotide sequences of genomic DNA which flank the borders of T-DNA integrated in the genome of plants. However, all the methods based on PCR have limitations which in some cases do not permit the desired objective to be achieved. We have developed a new technique for isolating flanking sequence tags (FSTs) via modified inverse PCR. This method is highly efficient and simple, but also retains the advantages of previously well-documented approaches.

  19. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    Energy Technology Data Exchange (ETDEWEB)

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong

    2011-12-31

    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  20. Biosynthetic porphyrins and the origin of photosynthesis

    Science.gov (United States)

    Mauzerall, D.; Ley, A.; Mercer-Smith, J. A.

    1986-01-01

    Since the prebiotic atmosphere was anaerobic, if not reducing, a useful function of primordial photosynthesis would have been to photooxidize reduced substrates such as Fe(+2), S(-2) or reduced organic molecules and to emit hydrogen. Experiments have shown that the early biogenic pigments uroporphyrin and coproporphyrin do photooxidize organic compounds and emit hydrogen in the presence of a platinum catalyst. These experiments were carried out in dilute aqueous solution near neutral pH under anaerobic atmosphere, and quantum yields near 10-2 were obtained. Thus relevant prebiotic conditions were maintained. Rather then to further optimize conditions, attempts were made to replace the platinum catalyst by a more prebiotically suitable catalyst. Trials with an Fe4S4(SR)4 cluster, in analogy to the present hydrogenase and nitrogenase, were not successful. However, experiments using cobalt complexes to catalyze the formation of hydrogen are promising. In analogy with biological photosynthetic systems which group pigments, electron transfer molecules and enzymes in clusters for efficiency, it was found that binding the biogenic porphyrins to the polyvinyl alcohol used to support the platinum catalyst did increase the quantum yield of the reaction. It was also found that ultraviolet light can serve to photo-oxidize porphyrinogens to porphyrins under anaerobic conditions. Thus the formation of the colorless porphyriogens by the extraordinarily simple biosynthetic pathway would not be a problem because of the prevalence of UV light in the prebiotic, anoxic atmosphere.

  1. 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.

  2. [Isolation of enterohemorrhagic Escherichia coli (O157:H7) by an immunomagnetic separation method].

    Science.gov (United States)

    Asai, Y; Murase, T; Osawa, R; Okitsu, T; Suzuki, R; Sata, S; Yamai, S; Wada, A; Tamura, K; Watanabe, H

    1997-01-01

    Three sporadic cases of enterohemorrhagic Escherichia coli (EHEC) O157 infection which occurred in Kanagawa in 1996 were investigated. In an attempt to determine sources of the infection, a novel method of immunomagnetic separation (IMS) was employed to isolate the bacterium from feces, foods, and other associated items. In the first case, strains of EHEC O157:H7 producing Vero toxin (VT) 2 were isolated from both feces of the patient and suspected food (cattle liver) kept at a restaurant, and the strains were found to be genotypically identical through an analysis of pulsed-field gel electrophoresis (PFGE). Subsequent investigation in the meat processing store, from which the above cattle liver had been retailed to the restaurants revealed that the store was contaminated with EHEC O157:H7 producing both VT1 and VT2. In the second case, a strain isolated from the patient was EHEC O157:H7 producing both VT1 and VT2 while strains isolated from the patient's family (without apparent symptom) and the suspected facility were O137:NM producing VT2. PFGE analysis indicated that the latter two strains were genotypically identical, suggesting that the facility thus contaminated with EHEC O157 caused the infection in question. In the third case, EHEC O157:NM producing VT2 was isolated from 4 out of 7 family members including the patient, and these strains were found to be genotypically identical by subsequent PFGE analysis. Source of the infection was, however, not determined due to lack of suspected food items. In this context, four slaughterhouses in Kanagawa Prefecture were investigated for presence of EHEC O157. As a result, strains of EHEC O157:H7 producing VT1 and VT2 were isolated from the contents of cattle's distal colon and surface of the skinned carcasses. Additional attempt was also made to determine a possibility of river water being contaminated with EHEC O157. The bacterium was, however, not isolated from water samples collected from 4 major rivers in the

  3. Deciphering the Late Biosynthetic Steps of Antimalarial Compound FR-900098

    OpenAIRE

    Johannes, Tyler W.; DeSieno, Matthew A.; Griffin, Benjamin M.; Thomas, Paul M.; Kelleher, Neil L.; Metcalf, William W.; Zhao, Huimin

    2010-01-01

    FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in E. coli by re-constructing the entire biosynthetic pathway using a three plasmid system. Based on this system, whole cell feeding assays in combination with in vitro enzymatic activity assays reveal an unprecedented functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These stud...

  4. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA

    OpenAIRE

    Wenli Li; Kui Hong; Weiming Zhu; Jingtao Zhang; Qiu Cui; Yuanyuan Du; Tong Li

    2013-01-01

    The indolocarbazole (ICZ) alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as...

  5. 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...

  6. Structures and comparative characterization of biosynthetic gene clusters for cyanosporasides, enediyne-derived natural products from marine actinomycetes

    OpenAIRE

    Lane, Amy L.; Nam, Sang Jip; Fukuda, Takashi; Yamanaka, Kazuya; Kauffman, Christopher A.; Jensen, Paul R; Fenical, William; Moore, Bradley S.

    2013-01-01

    Cyanosporasides are marine bacterial natural products containing a chlorinated cyclopenta[a]indene core of suspected enediyne polyketide biosynthetic origin. Herein, we report the isolation and characterization of novel cyanosporasides C–F (3–6) from the marine actinomycetes “Salinispora pacifica” CNS-143 and Streptomyces sp. CNT-179, highlighted by the unprecedented C-2' N-acetylcysteamine functionalized hexose group of 6. Cloning, sequencing, and mutagenesis of homologous ~50 kb cyanosporas...

  7. Design-based re-engineering of biosynthetic gene clusters: plug-and-play in practice

    OpenAIRE

    Frasch, Hans-Jörg; Medema, Marnix H.; Takano, Eriko; Breitling, Rainer; Gago, Federico; Parayil, Ajikumar

    2013-01-01

    Synthetic biology is revolutionizing the way in which the biosphere is explored for natural products. Through computational genome mining, thousands of biosynthetic gene clusters are being identified in microbial genomes, which constitute a rich source of potential novel pharmaceuticals. New methods are currently being devised to prioritize these gene clusters in terms of their potential for yielding biochemical novelty. High-potential gene clusters from any biological source can then be acti...

  8. Human insulin by tryptic transpeptidations of porcine insulin and biosynthetic precursors

    Energy Technology Data Exchange (ETDEWEB)

    Markussen, J.

    1987-01-01

    The combination of enzymatic semisynthesis with recombinant DNA techniques opens up new ways to tailor biosynthetic precursors in order to achieve the desired product, human insulin. The many factors which influence the course of these enzymatic processes have been studied systematically. This book, illustrated by 77 tables and 37 figures, reports these studies in detail. The book also reviews the thermodynamics behind enzymatic peptide synthesis, where methods are still being evolved.

  9. An improved method of mitochondrial DNA isolation for XL-PCR

    Institute of Scientific and Technical Information of China (English)

    SHI Duo; ZHU Ke-jun; WANG Xue-min; WANG Zhen-cheng; ZHENG Jian-ming; MIAO Ming-yong; JIAO Bing-hua

    2006-01-01

    Objective: To obtain high quality of mitochondrial DNA (mtDNA) and carry out extra-long PCR (XL-PCR). Methods: Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS (pH 7.0). mtDNA was then extracted using phenol and chloroform. Results: The mtDNA obtained by using our improved method can be used as effective template for XL-PCR,and total mtDNA (16 kb) can be amplified easily. Conclusion: Our improved method is effective in preparing high quality of mtDNA, which can be used as template for XL-PCR.

  10. Comparative identification of Candida species isolated from animals using phenotypic and PCR-RFLP methods

    Directory of Open Access Journals (Sweden)

    Nadăş George Cosmin

    2014-06-01

    Full Text Available The aim of this study was to identify 58 Candida sp. strains isolated from animals using the Chromatic Candida test, the API 20 C AUX system, and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP. The Chromatic Candida test was able to identify only C. albicans and C. krusei. The API 20 C AUX system and PCR-RFLP had similar specificity for the identification of Candida strains. In case of both methods, Candida albicans was the most frequently isolated species - 22 (37.93% strains, followed by Candida krusei - 17 (29.31% strains, Candida famata - 10 (17.24% strains, Candida parapsilosis - five (8.62% strains, and Candida kefyr - four (6.89% strains. PCR-RFLP represents a reliable, quick and relatively inexpensive genotyping method, recommended for rapid identification of Candida spp.

  11. A method for isolating and culturing placental cells from failed early equine pregnancies.

    Science.gov (United States)

    Rose, B V; Cabrera-Sharp, V; Firth, M J; Barrelet, F E; Bate, S; Cameron, I J; Crabtree, J R; Crowhurst, J; McGladdery, A J; Neal, H; Pynn, J; Pynn, O D; Smith, C; Wise, Z; Verheyen, K L P; Wathes, D C; de Mestre, A M

    2016-02-01

    Early pregnancy loss occurs in 6-10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14-65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL. PMID:26907389

  12. Activity of fosfomycin and comparison of several susceptibility testing methods against contemporary urine isolates.

    Science.gov (United States)

    Hirsch, Elizabeth B; Raux, Brian R; Zucchi, Paola C; Kim, Yisu; McCoy, Christopher; Kirby, James E; Wright, Sharon B; Eliopoulos, George M

    2015-12-01

    Fosfomycin is recommended as first-line treatment for acute uncomplicated cystitis in women. It has demonstrated in vitro activity against a variety of pathogens; however, a paucity of data are available from the USA. We determined the susceptibility of a collection of urine isolates to fosfomycin and compared multiple methods of susceptibility testing. Consecutive non-duplicate Enterobacteriaceae, enterococci and Pseudomonas aeruginosa isolates were collected from the clinical microbiology laboratory between August 2013 and January 2014. Isolates represented hospitalised or emergency department patients with monomicrobial bacteriuria. Fosfomycin MICs were determined in duplicate, on separate days, by Etest and disk diffusion and results were compared with agar dilution. Nitrofurantoin and ciprofloxacin were used as comparators. MIC results were categorised using Clinical and Laboratory Standards Institute interpretive criteria for Escherichia coli and Enterococcus faecalis. Correlation between the three testing methods was evaluated. Overall susceptibility to fosfomycin was 94.4%, 93.5% and 87.9% by agar dilution, disk diffusion and Etest, respectively. Five fosfomycin-resistant isolates were identified, including two Morganella morganii, one P. aeruginosa, one Proteus mirabilis and one Enterobacter aerogenes. Across all organisms, rates of essential agreement, categorical agreement, minor errors, major errors and very major errors for Etest/disk diffusion compared with agar dilution were 77.3%/NA, 89.5/93.8%, 7.1/5.0%, 3.6/1.3% and 0/0%, respectively. Fosfomycin displayed fairly consistent activity against a majority of isolates collected when using the susceptibility breakpoint of 64 μg/mL. MICs for E. coli were particularly low (≤2 μg/mL). These data lend support to current guidelines that recommend fosfomycin as empirical first-line therapy for uncomplicated UTI. PMID:26498988

  13. Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil

    OpenAIRE

    Nishizawa, Tomoyasu; Tago, Kanako; Uei, Yusuke; Ishii, Satoshi; Isobe, Kazuo; Otsuka, Shigeto; Senoo, Keishi

    2012-01-01

    We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution...

  14. An Aerodynamic Method for the Analysis of Isolated Horizontal-Axis Wind Turbines

    OpenAIRE

    Christian Masson; Idriss Ammara; Ion Paraschivoiu

    1997-01-01

    The aerodynamic analysis of a wind turbine represents a very complex task since it involves an unsteady three-dimensional viscous flow. In most existing performance-analysis methods, wind turbines are considered isolated so that interference effects caused by other rotors or by the site topology are neglected. Studying these effects in order to optimize the arrangement and the positioning of Horizontal-Axis Wind Turbines (HAWTs) on a wind farm is one of the research activities of the Bombardi...

  15. An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction

    OpenAIRE

    Ahmed, Zaheer; Fu, Yong-Bi

    2015-01-01

    Background Mitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substant...

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

    OpenAIRE

    M Johnson; Carpenter, E; Tian, Z.; Bruskiewich, R.; Burris, J.; Carrigan, C.; Chase, M; Clarke, N.; Covshoff, S.; Depamphilis, C.; Edger, P.; Goh, F.; Graham, S; S Greiner; Hibberd, J.

    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...

  17. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood

    OpenAIRE

    Tolios, Alexander; Teupser, Daniel; Lesca M Holdt

    2015-01-01

    Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated wi...

  18. Optimisation of a method for isolation of Clostridium difficile from faeces

    OpenAIRE

    Nilsson, Angelica

    2010-01-01

    Clostridium difficile is a pathogen for both humans and animals and is often associated with antibiotic-associated diarrhea. Recently, several human cases of C. difficile-infection with increased mortality and morbidity have been reported. In studies performed in different countries C. difficile has been found in meat. Therefore the question whether C. difficile can be a zoonotic agent has been raised. The aim of this study was to optimize a method for isolation of C. difficile from faeces. W...

  19. Isolation of radioactive iodothyronines for kinetic studies: a comparison of two methods

    International Nuclear Information System (INIS)

    A method based on the principle of gel separation followed by antibody extraction (GSAE) has been developed for isolation of radioactive thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3',5'-diiodothyronine (3',5'-T2) and 3'-monoiodothyronine (3'-T1) in serum. This method was used for the estimation of the metabolic clearance rate (MCR) of the iodothyronines using the single injection, non-compartmental approach, and was compared to the conventional trichloroacetic acid precipitation/ethanol extraction (TCA-E) technique. The GSAE method excluded the co-determination of radioactive iodine and iodoproetins, whereas the co-determination of radiolabelled daughter iodothyronines was found negligible. The relative difference of duplicate estimations of MCR was approximately 10%. Using the TCA-E method for isolation of tracer, the MCR of T4, T3 and rT3 was underestimated to a minor degree (20%), whereas the MCRs of 3,3'-T2, 3',5'-T2 and 3'-T1 were 20-40% of those estimated by the GSAE method In conclusion the GSAE method was found suitable for kinetic studies of iodothyronines, whereas the TCA-E method cannot be used for turnover studies of 3,3'-T2, 3',5'-T2 or 3'-T1. (author)

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

    International Nuclear Information System (INIS)

    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

  1. Origin of saxitoxin biosynthetic genes in cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Ahmed Moustafa

    Full Text Available BACKGROUND: Paralytic shellfish poisoning (PSP is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX. STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. METHODOLOGY/PRINCIPAL FINDINGS: We generated a draft genome assembly of the saxitoxin-producing (STX+ cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX- sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena

  2. Simple Method for Detection of Metallo – β–Lactamase Among Gram Negative Isolates

    Directory of Open Access Journals (Sweden)

    Agrawal R

    2015-10-01

    Full Text Available Background: Carbapenem resistance due to the production of metallo-β -lactamase (MBL in Gram-negative organism is an increasing public health problem. Aim: The purpose of this study is to detectMBL in Gram Negative bacterial isolates among Ventilator Associated Pneumonia patients. Materials and Methods: Phenotypic detection of MBL was done by three methods: 1.Modified Hodge Test (MHT 2.Combined disc test (CDT 3.Double Disc Synergy Test (DDST. Results: Out of 126 gram negative bacterial isolates, 80 (63.49% showed resistance to carbapenem group of drugs. Among them maximum resistance was shown by Acinetobacter baumanii (90.32%, followed by Klebseilla pneumoniae (45.7%, Pseudomonas aeruginosa (26%. Out of 80 isolates, 66 were positive for MBL by MHT, 64 by CDT and 61 were detected positive for MBL by DDST. Conclusion: MHT and CDT were found equally efficient method to detect MBL. Maximum MBL production was detected in Acinetobacter baumanii. Simple and accurate screening test is required to prevent the spread of nosocomial strain in hospitals.

  3. An improved method for isolation of epithelial and stromal cells from the human endometrium.

    Science.gov (United States)

    Masuda, Ayako; Katoh, Noriko; Nakabayashi, Kazuhiko; Kato, Kiyoko; Sonoda, Kenzo; Kitade, Mari; Takeda, Satoru; Hata, Kenichiro; Tomikawa, Junko

    2016-04-22

    We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from the human endometrium. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease treatment and cell suspension conditions to dissociate single cells from the tissue fragments and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 × 10(6) of EMECs and 2.8 × 10(6) EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the molecular mechanisms underlying the dynamic changes of the endometrium during the menstrual cycle. PMID:26853786

  4. LASER BIOLOGY: Peculiarities of studying an isolated neuron by the method of laser interference microscopy

    Science.gov (United States)

    Yusipovich, Alexander I.; Novikov, Sergey M.; Kazakova, Tatiana A.; Erokhova, Liudmila A.; Brazhe, Nadezda A.; Lazarev, Grigory L.; Maksimov, Georgy V.

    2006-09-01

    Actual aspects of using a new method of laser interference microscopy (LIM) for studying nerve cells are discussed. The peculiarities of the LIM display of neurons are demonstrated by the example of isolated neurons of a pond snail Lymnaea stagnalis. A comparative analysis of the images of the cell and subcellular structures of a neuron obtained by the methods of interference microscopy, optical transmission microscopy, and confocal microscopy is performed. Various aspects of the application of LIM for studying the lateral dimensions and internal structure of the cytoplasm and organelles of a neuron in cytology and cell physiology are discussed.

  5. [Identification of filamentous fungi isolated from clinical samples by two different methods and their susceptibility results].

    Science.gov (United States)

    Direkel, Sahin; Otağ, Feza; Aslan, Gönül; Ulger, Mahmut; Emekdaş, Gürol

    2012-01-01

    Molds are widely distributed in nature. Aspergillus spp. represent the most frequently observed causative agents, however less frequent pathogens Fusarium, Scedosporium and Zygomycetes have also been considered the most important causes of morbidity and mortality in profoundly immunosuppressed hosts. The aims of this study were to identify filamentous fungi isolated from clinical specimens by conventional and molecular methods, and to detect their antifungal susceptibilities. A total of 6742 clinical specimens obtained from hospitalized patients at critical units of Mersin University Medical Faculty Hospital and sent to our laboratory between April 2008-January 2010 were included in the study. The isolates were identified by classical mycological methods and polymerase chain reaction-based DNA sequencing. Susceptibilities to fluconazole and voriconazole were tested by disk diffusion method and to fluconazole, voriconazole, amfoterisin B, caspofungin and posaconazole by E-test. Filamentous fungi were isolated from 71 (1.05%) samples (13 sputum, 4 wound, 4 peritoneal fluid, 3 extrenal ear discharge, 3 abscess and one of each cerebrospinal fluid, blood, tissue biopsy, nasal swab and conjunctival swab) which belonged to 32 patients (13 female, 19 male; age range 7 months-77 years, mean age: 46.6 years). Of the patients 62.3% presented one or more risk factors such as chronic renal failure (n= 8), chronic obstructive lung disease (n= 6), malignancy (n= 6), diabetes mellitus (n= 5) and peripheral vascular disease (n= 5). Of the isolates six were identified as Aspergillus niger, six as Aspergillus flavus, five as Aspergillus fumigatus, four as Aspergillus terreus, five as Fusarium spp., two as Bipolaris spp., and one of each as Acremonium spp., Aurebasidium spp., Mucor spp., and Scedosporium spp. By conventional methods. Three isolates exhibited different identities by DNA sequencing. All Aspergillus isolates were correctly identified at species level by both methods

  6. Detection of Anthropogenic Particles in Fish Stomachs: An Isolation Method Adapted to Identification by Raman Spectroscopy.

    Science.gov (United States)

    Collard, France; Gilbert, Bernard; Eppe, Gauthier; Parmentier, Eric; Das, Krishna

    2015-10-01

    Microplastic particles (MP) contaminate oceans and affect marine organisms in several ways. Ingestion combined with food intake is generally reported. However, data interpretation often is circumvented by the difficulty to separate MP from bulk samples. Visual examination often is used as one or the only step to sort these particles. However, color, size, and shape are insufficient and often unreliable criteria. We present an extraction method based on hypochlorite digestion and isolation of MP from the membrane by sonication. The protocol is especially well adapted to a subsequent analysis by Raman spectroscopy. The method avoids fluorescence problems, allowing better identification of anthropogenic particles (AP) from stomach contents of fish by Raman spectroscopy. It was developed with commercial samples of microplastics and cotton along with stomach contents from three different Clupeiformes fishes: Clupea harengus, Sardina pilchardus, and Engraulis encrasicolus. The optimized digestion and isolation protocol showed no visible impact on microplastics and cotton particles while the Raman spectroscopic spectrum allowed the precise identification of microplastics and textile fibers. Thirty-five particles were isolated from nine fish stomach contents. Raman analysis has confirmed 11 microplastics and 13 fibers mainly made of cellulose or lignin. Some particles were not completely identified but contained artificial dyes. The novel approach developed in this manuscript should help to assess the presence, quantity, and composition of AP in planktivorous fish stomachs. PMID:26289815

  7. Methods and apparatus using commutative error detection values for fault isolation in multiple node computers

    Science.gov (United States)

    Almasi, Gheorghe [Ardsley, NY; Blumrich, Matthias Augustin [Ridgefield, CT; Chen, Dong [Croton-On-Hudson, NY; Coteus, Paul [Yorktown, NY; Gara, Alan [Mount Kisco, NY; Giampapa, Mark E [Irvington, NY; Heidelberger, Philip [Cortlandt Manor, NY; Hoenicke, Dirk I [Ossining, NY; Singh, Sarabjeet [Mississauga, CA; Steinmacher-Burow, Burkhard D [Wernau, DE; Takken, Todd [Brewster, NY; Vranas, Pavlos [Bedford Hills, NY

    2008-06-03

    Methods and apparatus perform fault isolation in multiple node computing systems using commutative error detection values for--example, checksums--to identify and to isolate faulty nodes. When information associated with a reproducible portion of a computer program is injected into a network by a node, a commutative error detection value is calculated. At intervals, node fault detection apparatus associated with the multiple node computer system retrieve commutative error detection values associated with the node and stores them in memory. When the computer program is executed again by the multiple node computer system, new commutative error detection values are created and stored in memory. The node fault detection apparatus identifies faulty nodes by comparing commutative error detection values associated with reproducible portions of the application program generated by a particular node from different runs of the application program. Differences in values indicate a possible faulty node.

  8. Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus.

    Science.gov (United States)

    Zhu, Xiaoxuan; Zeng, Xinyi; Sun, Chao; Chen, Shilin

    2014-09-01

    Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids (TIAs) in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology. PMID:25159992

  9. Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.

    Science.gov (United States)

    Rood, Ilse M; Deegens, Jeroen K J; Merchant, Michael L; Tamboer, Wim P M; Wilkey, Daniel W; Wetzels, Jack F M; Klein, Jon B

    2010-10-01

    Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome. PMID:20686450

  10. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms

    Science.gov (United States)

    Wang, Yan; Wang, Liuqing; Liu, Fei; Wang, Qi; Selvaraj, Jonathan Nimal; Xing, Fuguo; Zhao, Yueju; Liu, Yang

    2016-01-01

    Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms. PMID:27007394

  11. Improved Method for Isolation of Microbial RNA from Biofuel Feedstock for Metatranscriptomics

    Energy Technology Data Exchange (ETDEWEB)

    Piao, Hailan; Markillie, Lye Meng; Culley, David E.; Mackie, Roderick I.; Hess, Matthias

    2013-03-28

    Metatranscriptomics—gene express profiling via DNA sequencing—is a powerful tool to identify genes that are ac- tively expressed and might contribute to the phenotype of individual organisms or the phenome (the sum of several phenotypes) of a microbial community. Furthermore, metatranscriptome studies can result in extensive catalogues of genes that encode for enzymes of industrial relevance. In both cases, a major challenge for generating a high quality metatranscriptome is the extreme lability of RNA and its susceptibility to ubiquitous RNAses. The microbial commu- nity (the microbiome) of the cow rumen efficiently degrades lignocelullosic biomass, generates significant amounts of methane, a greenhouse gas twenty times more potent than carbon dioxide, and is of general importance for the physio- logical wellbeing of the host animal. Metatranscriptomes of the rumen microbiome from animals kept under different conditions and from various types of rumen-incubated biomass can be expected to provide new insights into these highly interesting phenotypes and subsequently provide the framework for an enhanced understanding of this socio- economically important ecosystem. The ability to isolate large amounts of intact RNA will significantly facilitate accu- rate transcript annotation and expression profiling. Here we report a method that combines mechanical disruption with chemical homogenization of the sample material and consistently yields 1 mg of intact RNA from 1 g of rumen-in- cubated biofuel feedstock. The yield of total RNA obtained with our method exceeds the RNA yield achieved with pre- viously reported isolation techniques, which renders RNA isolated with the method presented here as an ideal starting material for metatranscriptomic analyses and other molecular biology applications that require significant amounts of starting material.

  12. Isolation of Staphylococcus aureus from raw fish in relation to culture methods.

    Science.gov (United States)

    Saito, Etsuko; Yoshida, Nanako; Kawano, Junichi; Shimizu, Akira; Igimi, Shizunobu

    2011-03-01

    Five hundred and fifty fish samples from various stages in the course of distribution in Hyogo Prefecture (209 retailed in super markets, 173 obtained from fishery cooperatives at a harbor, 91 caught by trawling and 77 caught by rod fishing) were examined for contamination with Staphylococcus aureus (S. aureus). S. aureus was detected in 41 (19.6%) of the retail fish samples and 46 (26.6%) of the samples from the fishery cooperatives. No S. aureus was isolated from the live fish (91 trawled and 77 fished by rod). With regard to the retail fish, the contamination rate of processed fish (26.0%) was significantly higher than that of unprocessed fish (14.2%). For 88 samples, the efficacy of the selective medium was compared using Baird-Parker agar and mannitol salt agar supplemented with egg yolk (MSEY agar) by the direct plate and enrichment culture methods. Using the direct culture method, the S. aureus positive rate with the Baird-Parker agar (30.7%) was significantly higher (Pagar (6.8%). The enrichment culture method remarkably raised the S. aureus detection rate. Seventy-eight (85.7%) of 91 isolates belonged to the human ecovar. Sixty-two (68.1%) of the 91 isolates had some enterotoxin genes, including 44 (48.4%) with the sea gene. These data showed that the fish were contaminated with S. aureus after landing and that Baird-Parker agar had an advantage in detecting S. aureus with a direct plate culture. PMID:20953131

  13. 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

  14. Novel Simplified and Rapid Method for Screening and Isolation of Polyunsaturated Fatty Acids Producing Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Ashwini Tilay

    2012-01-01

    Full Text Available Bacterial production of polyunsaturated fatty acids (PUFAs is a potential biotechnological approach for production of valuable nutraceuticals. Reliable method for screening of number of strains within short period of time is great need. Here, we report a novel simplified method for screening and isolation of PUFA-producing bacteria by direct visualization using the H2O2-plate assay. The oxidative stability of PUFAs in growing bacteria towards added H2O2 is a distinguishing characteristic between the PUFAs producers (no zone of inhibition and non-PUFAs producers (zone of inhibition by direct visualization. The confirmation of assay results was performed by injecting fatty acid methyl esters (FAMEs produced by selected marine bacteria to Gas Chromatography-Mass Spectrometry (GCMS. To date, this assay is the most effective, inexpensive, and specific method for bacteria producing PUFAs and shows drastically reduction in the number of samples thus saves the time, effort, and cost of screening and isolating strains of bacterial PUFAs producers.

  15. 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.

  16. Contribution to the study of radioactive ion beam production by the ISOL method

    International Nuclear Information System (INIS)

    This thesis is related to the R and D program for the production of radioactive ion beams by the ISOL method at GANIL in Caen. This work concerns several different techniques based on the ISOL method. The first one is the production of radioactive ion beams with a SPIRAL target-source system (target + ECR source). The production rates of radioactive neon beams were determined on the SIRa test bench and previsions for SPIRAL were established. The feasibility of the production of radioactive condensable element beams with such target-source system, using a transport under a volatile molecular form between the target and the source, was experimentally proven by the production of radioactive oxygen beams via the CO molecule. The second technique is the production of radioactive alkaline beams with the target-source system MONOLITHE (target + hot cavity source). The production efficiencies of lithium and sodium radioactive beams were determined. A new methodology, the 'global method', has been developed as part of this thesis, for deducing diffusion, effusion and ionisation properties of these two elements with this ensemble. It is shown that the evolution of diffusion properties between different alkali elements is similar to noble gases. The third one is the IGISOL technique (target + ion guide). The MI-GI-CHEMIN code was created for simulating the movement of ions in an ion guide filled with helium and a given concentration of impurities, including electric and magnetic fields. A first IGISOL prototype is in realisation at GANIL. (author)

  17. Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LI Dong-rui; CAI Jian-hui

    2012-01-01

    Objective This literature review aims to summarize the methods of isolation,expansion,differentiation and preservation of human umbilical cord mesenchymal stem cells (hUCMSCs),for comprehensive understanding and practical use in preclinical research and clinical trials.Data sources All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure (CNKI).Studies were retrieved using the key word "human umbilical cord mesenchymal stem cells".Results Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods.Culture conditions may affect the expansion and differentiating orientations of hUCMSCs.In addition,hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed.Conclusion Considering their multi-potential,convenient and non-invasive accessibility,low immunogenicity and the reported therapeutic effects in several different preclinical animal models,hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

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

    International Nuclear Information System (INIS)

    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)

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

    Directory of Open Access Journals (Sweden)

    Michael D Barton

    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

  20. 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.

  1. A method for isolating smooth muscle cells from pig urinary bladder with low concentrations of collagenase and papain: the relation between calcium concentration and isolated cell length.

    NARCIS (Netherlands)

    E. van Asselt (Els); R. van Mastrigt (Ron); R. Schot

    1993-01-01

    textabstractThe present study describes a method for isolating single smooth muscle cells from pig urinary bladder using a continuous resuspension device. Low concentrations of collagenase and papain were sufficient to obtain a high yield of viable smooth muscle cells, which remained viable for abou

  2. Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Luciano Mengatto

    2006-08-01

    Full Text Available A study was carried out to compare the performance of a commercial method (MGIT and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA, microscopic observation drug susceptibility (MODS assay, MTT test, and broth microdilution method (BMM. A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM was used as gold standard. MGIT, NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90%. With most of the assays, sensitivity for ethambutol was low (62-87% whereas for streptomycin, sensitivity values ranged from 84 to 100%; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM were seen with isolates whose minimal inhibitory concentrations fell close the cutoff. MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8; more standardization is needed for ethambutol.

  3. The evaluation of susceptibility of clinical and environmental Nontuberculosis mycobacterium isolated from Isfahan to Ciprofloxacin by Agar dilution method

    Directory of Open Access Journals (Sweden)

    Tooba Radaei

    2013-01-01

    Full Text Available Introduction: Ciprofloxacin is a fluoroquinolone antibiotic which is active against mycobacteria and functions by inhibiting DNA gyrase and topoisomerase IV enzymes. Resistance to ciprofloxacin and other fluoroquinolones may evolve rapidly, even during a course of treatment. Nowadays, mycobacteria exhibit resistance worldwide and usage of the fluoroquinolones, particularly in nontuberculous mycobacteria disease, has complicated the related treatments. Materials and methods: A total of 39 clinical and environmental isolates of NTM from microbial collections of Isfahan Microbiology Department and Tuberculosis center were obtained. The isolates were investigated by primary conventional methods consisting of colony characteristics, pigmentation, growth temperature, rate of growth and Ziehl–Neelsen staining. The susceptibility of isolates to the concentrations of 1, 2 and 4 µg/ml of ciprofloxacin was determined by agar dilution method according to the CLSI guideline.Results: Thirty nine isolates were identified by phenotypic tests. The frequency of isolates was as follow: M. fortuitum; 25 cases, M. gordonae; 10 cases, M. smegmatis; 1 case, M.‏conceptionense; 1 case and M. abscessus; 2 cases. All isolates except Mycobacterium abscessus were sensitive to all three concentrations of 1, 2 and 4 µg/ml ciprofloxacin.Discussion and conclusion: Due to the sensitivity of environmental nontuberculous mycobacteria isolates (except M. abscessus and clinical isolates including M. fortuitum and M.‏gordonae to ciprofloxacin, this antibiotic could be regarded as the original drug in the treatment of these infections.

  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. A method for detergent-free isolation of membrane proteins in their local lipid environment.

    Science.gov (United States)

    Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow, Rosemary A; Lin, Yu-Pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench, Stephen P; Dafforn, Timothy R

    2016-07-01

    Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins. PMID:27254461

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

    International Nuclear Information System (INIS)

    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/m2 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

  7. A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate.

    Science.gov (United States)

    Tong, Chenyao; Geng, Fang; He, Zhenjiao; Cai, Zhaoxia; Ma, Meihu

    2015-01-01

    Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4°C before being centrifuged at 4°C. The resulting supernatant was added to 35% (w/v) (NH4)2SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale. PMID:25542196

  8. Comparison of methods for the isolation of human breast epithelial and myoepithelial cells

    Directory of Open Access Journals (Sweden)

    Arantzazu eZubeldia-Plazaola

    2015-05-01

    Full Text Available Two lineages, epithelial and myoepithelial cells, are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells and CD10/K14 (myoepithelial cells antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast

  9. Comparison of methods for the isolation of human breast epithelial and myoepithelial cells.

    Science.gov (United States)

    Zubeldia-Plazaola, Arantzazu; Ametller, Elisabet; Mancino, Mario; Prats de Puig, Miquel; López-Plana, Anna; Guzman, Flavia; Vinyals, Laia; Pastor-Arroyo, Eva M; Almendro, Vanessa; Fuster, Gemma; Gascón, Pedro

    2015-01-01

    Two lineages, epithelial, and myoepithelial cells are the main cell populations in the normal mammary gland and in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures obtained from fresh breast tissue are a powerful tool to study more reliably new aspects of mammary gland biology, including normal and pathological conditions. Nevertheless, the methods described to date have some technical problems in terms of cell viability and yield, which hamper work with primary mammary cells. Therefore, there is a need to optimize technology for the proper isolation of epithelial and myoepithelial cells. For this reason, we compared four methods in an effort to improve the isolation and primary cell culture of different cell populations of human mammary epithelium. The samples were obtained from healthy tissue of patients who had undergone mammoplasty or mastectomy surgery. We based our approaches on previously described methods, and incorporated additional steps to ameliorate technical efficiency and increase cell survival. We determined cell growth and viability by phase-contrast images, growth curve analysis and cell yield, and identified cell-lineage specific markers by flow cytometry and immunofluorescence in 3D cell cultures. These techniques allowed us to better evaluate the functional capabilities of these two main mammary lineages, using CD227/K19 (epithelial cells) and CD10/K14 (myoepithelial cells) antigens. Our results show that slow digestion at low enzymatic concentration combined with the differential centrifugation technique is the method that best fits the main goal of the present study: protocol efficiency and cell survival yield. In summary, we propose some guidelines to establish primary mammary epithelial cell lines more efficiently and to provide us with a strong research instrument to better understand the role of different epithelial cell types in the origin of breast cancer. PMID

  10. 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.

  11. Identification of a Pantoea biosynthetic cluster that directs the synthesis of an antimicrobial natural product.

    Science.gov (United States)

    Walterson, Alyssa M; Smith, Derek D N; Stavrinides, John

    2014-01-01

    Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea. PMID:24796857

  12. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    NARCIS (Netherlands)

    Pham, T.H.T.

    2013-01-01

      The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random

  13. 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.

  14. Refinement of the manumycin-type antibiotics biosynthetic model

    Czech Academy of Sciences Publication Activity Database

    Petříček, Miroslav; Yu, T.-W.; Pospíšil, Stanislav; Petříčková, Kateřina; Krištůfek, Václav

    Newcastle upon Tyne: International Commitee, UK body, University of Newcastle upon Tyne, 2007. s. 167. [International Symposium on the Biology of Actinomycetes /14./. 26.08.2007-30.08.2007, The Sage Gateshead] Institutional research plan: CEZ:AV0Z60660521; CEZ:AV0Z50200510 Keywords : manumycin-type antibiotics * biosynthetic model Subject RIV: EH - Ecology, Behaviour

  15. Variation in the Trichothecene Mycotoxin Biosynthetic Gene Cluster in Fusarium

    Science.gov (United States)

    Trichothecene mycotoxins are produced by some plant pathogenic species of the fungus Fusarium and can contribute to its virulence on some plants. In Fusarium graminearum and F. sporotrichioides trichothecene biosynthetic enzymes are encoded at three loci: the single-gene TRI101 locus; the two-gene ...

  16. 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...

  17. Improved method for isolation of coupled mitochondria of Araucaria angustifolia (Bert. O. Kuntze

    Directory of Open Access Journals (Sweden)

    André Bellin Mariano

    2004-11-01

    Full Text Available A method for the isolation of coupled mitochondria from the callus of Araucaria angustifolia is described for the first time. Mitochondria were isolated from embryogenic callus of A. angustifolia. They were metabolically active, able to sustain oxidative phosphorylation as shown by respiratory control ratio values, which were about 2.4 when respiring on succinate as substrate. Oxygen uptake experiments, using freeze-thawed disrupted mitochondria, showed the presence of alternative rotenone-insensitive NAD(PH dehydrogenases, which were stimulated by Ca2+. The procedure now described for the isolation of A. angustifolia mitochondria is an important new tool, allowing the investigation of mitochondrial bioenergetics and metabolism and physiology of plants.Um procedimento de isolamento de mitocôndrias funcionalmente intactas de calos embriogênicos de Araucaria angustifolia foi desenvolvido pela primeira vez em nosso laboratório. Mitocôndrias isoladas por este método são metabolicamente ativas, capazes de sustentar fosforilação oxidativa como mostrado pelo controle respiratório de aproximadamente 2,4, respirando na presença de succinato como substrato. Através de experimentos de consumo de oxigênio com mitocôndrias rompidas em nitrogênio líquido foi demonstrada a presença de NAD(PH desidrogenases alternativas, insensíveis à rotenona e estimuladas por Ca2+. O isolamento de mitocôndrias de A. angustifolia é um novo e importante instrumento para estudar plantas, permitindo a execução de múltiplas investigações a respeito da bioenergética mitocondrial e fisiologia vegetal.

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

    International Nuclear Information System (INIS)

    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

  19. 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.

  20. Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes.

    Directory of Open Access Journals (Sweden)

    Marc T J Johnson

    Full Text Available 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.

  1. Intercomparison of analysis methods for seismically isolated nuclear structures (KAERI HLRB and CRIEPI Isolated Rigid Mass Mock-Up)

    International Nuclear Information System (INIS)

    The combined shear and compression behaviors of the KAERI HLRB made of MRPRA rubber and the shaking responses of the CRIEPI isolated rigid mass mock-up are analyzed. For FEM analyses of KAERI HLRB, three kinds of strain energy density functions of the ABAQUS program are used as constitutive law for rubber with hyperelastic characteristics. The analysis results are compared with test results, depending on the constitutive models. The simulation results for the shaking table tests of the CRIEPI rigid mass mock-up supported by scaled lead rubber bearings are obtained by ABAQUS time history analyses. In the analysis, the linear and bilinear hysterisis models simulating the behaviors of the rubber bearing are used. (author)

  2. 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

    2012-01-01

    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. PMID:22653584

  3. 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. PMID:26805977

  4. 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

  5. An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

    OpenAIRE

    Lorenz, W Walter; Yu, Yuan-Sheng; Dean, Jeffrey F. D.

    2010-01-01

    Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for mic...

  6. 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

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

    International Nuclear Information System (INIS)

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [32P]-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

  8. 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. PMID:27474618

  9. A Novel Method for Screening Children with Isolated Bicuspid Aortic Valve.

    Science.gov (United States)

    Gharehbaghi, Arash; Dutoit, Thierry; Sepehri, Amir A; Kocharian, Armen; Lindén, Maria

    2015-12-01

    This paper presents a novel processing method for heart sound signal: the statistical time growing neural network (STGNN). The STGNN performs a robust classification by merging supervised and unsupervised statistical methods to overcome non-stationary behavior of the signal. By combining available preprocessing and segmentation techniques and the STGNN classifier, we build an automatic tool for screening children with isolated BAV, the congenital heart malformation which can lead to serious cardiovascular lesions. Children with BAV (22 individuals) and healthy condition (28 individuals) are subjected to the study. The performance of the STGNN is compared to that of a time growing neural network (CTGNN) and a conventional support vector (CSVM) machine, using balanced repeated random sub sampling. The average of the accuracy/sensitivity is estimated to be 87.4/86.5 for the STGNN, 81.8/83.4 for the CTGNN, and 72.9/66.8 for the CSVM. Results show that the STGNN offers better performance and provides more immunity to the background noise as compared to the CTGNN and CSVM. The method is implementable in a computer system to be employed in primary healthcare centers to improve the screening accuracy. PMID:26577485

  10. 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. PMID:22083917

  11. Mechanical properties and design method of thick rubber bearings for three dimensional base isolation

    International Nuclear Information System (INIS)

    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. 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 lading conditions. From the test results, we confirm that full scale thick rubber bearing to satisfy requirements is feasible. Furthermore, 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. (author)

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

    International Nuclear Information System (INIS)

    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)

  13. Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

    Science.gov (United States)

    Baranyai, Tamás; Herczeg, Kata; Onódi, Zsófia; Voszka, István; Módos, Károly; Marton, Nikolett; Nagy, György; Mäger, Imre; Wood, Matthew J.; El Andaloussi, Samir; Pálinkás, Zoltán; Kumar, Vikas; Nagy, Péter; Kittel, Ágnes; Buzás, Edit Irén; Ferdinandy, Péter; Giricz, Zoltán

    2015-01-01

    Background Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. Aim Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). Methods and Results Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. Conclusion Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield. PMID:26690353

  14. Cyclic Lipopeptide Biosynthetic Genes and Products, and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria.

    Directory of Open Access Journals (Sweden)

    Isabel Mora

    Full Text Available The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP biosynthetic genes ituC (iturin, bmyB (bacillomycin, fenD (fengycin and srfAA (surfactin, and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP genes were bmyB, srfAA and fenD (34-50% of isolates. Most isolates (98.4% produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the

  15. Cyclic Lipopeptide Biosynthetic Genes and Products, and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria.

    Science.gov (United States)

    Mora, Isabel; Cabrefiga, Jordi; Montesinos, Emilio

    2015-01-01

    The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of

  16. Evaluation of Methods for AmpC Beta-Lactamase in Gram Negative Clinical Isolates from Tertiary Care Hospitals

    OpenAIRE

    Singhal S; Mathur T; Khan S; Upadhyay D; Chugh S; Gaind R; Rattan A

    2005-01-01

    The purpose of this study was to simultaneously screen for Extended-spectrum b-lactamases (ESBL) and AmpC b-lactamases in gram negative clinical isolates from four tertiary care hospitals and further to compare two detection methods three-dimensional extraction method and AmpC disk test for AmpC b-lactamases. A total of 272 isolates were screened for ESBL and AmpC β-lactamase by modified double disk approximation method (MDDM). Synergy observed between disks of ceftazidime/cefotaxime a...

  17. 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

  18. Mutagenesis as a Functional Genomics Platform for Pharmaceutical Alkaloid Biosynthetic Gene Discovery in Opium Poppy

    International Nuclear Information System (INIS)

    Opium poppy (Papaver somniferum) accumulates the analgesic benzyl-isoquinoline alkaloids morphine, codeine and thebaine, and remains one of the world's most important medicinal plants. The development of varieties that accumulate valuable compounds, such as thebaine and codeine, but not morphine precludes the illicit synthesis of heroin (O,O-diacetylmorphine) and has led to the establishment of alternative cash crops. Novel cDNAs encoding a growing number of biosynthetic enzymes have been isolated, and various -omics resources including EST databases and DNA microarray chips have been established. However, the full potential of functional genomics as a tool for gene discovery in opium poppy remains limited by the relative inefficiency of genetic transformation protocols, which also restricts the application of metabolic engineering for both experimental and commercial purposes. We are establishing an effective functional genomics initiative based on induced mutagenesis and recently developed reverse genetics methodology, such as TILLING (Targeting Induced Local Lesions IN Genomes), with the aim of identifying biosynthetic genes that can be used to engineer opium poppy for the production of copious levels of high-value pharmaceutical alkaloids. Mutagenesis involves the treatment of seeds with ethyl methane sulfonate (EMS) or by fast-neutron bombardment (FNB). In preliminary experiments with EMS-treated seeds, the screening of 1,250 independent M2 plants led to the isolation of four mutants that displayed two distinctly altered alkaloid profiles. Two lines accumulated the central pathway intermediate reticuline and relatively low levels of morphine, codeine and thebaine compared to wild-type plants. Two other lines showed the unusual accumulation in the latex of the antimicrobial alkaloid sanguinarine, which is the product of a branch pathway distinct from that leading to morphine. The present status of -omics resources and functional genomics platforms available to

  19. Mutagenesis as a functional genomics platform for pharmaceutical alkaloid biosynthetic gene discovery in opium poppy

    International Nuclear Information System (INIS)

    Opium poppy (Papaver somniferum) accumulates the analgesic alkaloids morphine, codeine and thebaine, and remains one of the world's most important medicinal plants. The development of varieties that accumulate valuable compounds, such as thebaine and codeine, but not morphine precludes the illicit synthesis of heroin (O,O-diacetylmorphine) and has created opportunities to establish alternative cash crops. Novel cDNAs encoding more than a dozen biosynthetic enzymes have been isolated, and substantial EST databases and DNA microarray chips have been established. The full potential of functional genomics as a tool for gene discovery in opium poppy remains limited by the relative inefficiency of genetic transformation protocols, which also restricts the application of metabolic engineering for both experimental and commercial purposes. We are establishing an effective functional genomics initiative based on induced mutagenesis and TILLING (Targeting Induced Local Lesions IN Genomes) and with the aim of identifying biosynthetic genes that can be used to engineer opium poppy to produce copious levels of high-value pharmaceutical alkaloids. Mutagenesis involves the treatment of seeds by fast-neutron bombardment (FNB) or with ethyl methane sulfonate (EMS). Mutagenized opium poppy plants are cultivated in a secure underground growth facility in partnership with a Canadian biotechnology company. In preliminary experiments with EMS-treated seeds, the screening of 1,250 independent M2 plants led to the isolation of four mutants that displayed two distinctly altered alkaloid profiles. Two lines accumulated the central pathway intermediate (S)- reticuline and only low levels of morphine, codeine and thebaine. Two other lines showed the unusual accumulation of the antimicrobial alkaloid sanguinarine, which is the product of a branch pathway distinct from that leading to morphine, in the latex. The present status of -omics resources and functional genomics platforms available to

  20. Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces.

    Science.gov (United States)

    Harmon, Aaron F; Zarlenga, Dante S; Hildreth, Michael B

    2006-02-18

    A multiplex PCR assay for differentiating strongyle eggs from cattle has recently been described; however, the egg disruption and DNA extraction procedures, though effective, are inadequate for large studies or clinical application. The purpose of this research was to evaluate methods for disrupting trichostrongyle eggs, then assess commercial kits for extracting egg DNA using Ostertagia ostertagi as a model species. Egg disruption procedures tested included probe sonication, bath sonication, bead beating, boiling, microwaving, proteinase K/SDS digestion, freezing, and various combinations of the above with the incorporation of sodium dodecyl sulfate. These procedures were evaluated in conjunction with four commercial DNA extraction kits: DNA Stool mini kit and DNeasy Plant kit (Qiagen), Fast DNA kit (QBiogene), and the MAP extraction kit (Tetracore). Results showed that egg disruption was best accomplished with the bead beater and ceramic beads, resulting in 100% disruption within 1min. When DNA extraction was preceded by the isolation of eggs from feces, all procedures except the Fast DNA kit produced PCR-ready DNA from at least two eggs. The DNeasy Plant kit allowed consistent detection of DNA released from one egg. Due to the morphological similarities among trichostrongyle eggs in ruminants, strongyle eggs in equids, and hookworm eggs, the methods described herein may have broad application to other nematodes. PMID:16303253

  1. Isolation valve leakage: an overview of U.S. methods for evaluation and mitigation

    International Nuclear Information System (INIS)

    Isolation valve leakage has resulted in cracking in various piping systems at several nuclear power plants worldwide, beginning with the Farley, Tihange and Genkai incidents in 1987-1988, and more recently the incidents at Dampierre. The phenomena associated with thermal stratification induced by leakage are diverse and complicated due to the wide variety of geometric and thermal-hydraulic conditions encountered in reactor piping systems. To understand the mechanisms that lead to these phenomena and to develop strategies to mitigate their consequences, methods have been developed in the US for predicting and evaluating thermal loads related to thermal stratification. These methods include the determination of the stratification interface height from the bottom inner surface of the pipe, the extent of piping that is heated by header pipe flow, the extent of branch piping that may experience thermal cycling induced by header pipe flow turbulence, and the heating of a leakage flow in sections of branch piping near the header pipe. This methodology may be used to postulate thermal loadings in order to evaluate the effects of leakage on stress and fatigue, to determine preferred temperature monitoring locations, to improve inspection programs, or to design piping systems resistant to cracking for new plants. (authors)

  2. Cloning, Characterization and Heterologous Expression of the Indolocarbazole Biosynthetic Gene Cluster from Marine-Derived Streptomyces sanyensis FMA

    Directory of Open Access Journals (Sweden)

    Wenli Li

    2013-02-01

    Full Text Available The indolocarbazole (ICZ alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as a probe to isolate the 34.6-kb DNA region containing the spc gene cluster. Sequence analysis revealed genes for ICZ ring formation (spcO, D, P, C, sugar unit formation (spcA, B, E, K, J, I, glycosylation (spcN, G, methylation (spcMA, MB, as well as regulation (spcR. Their involvement in ICZ biosynthesis was confirmed by gene inactivation and heterologous expression in Streptomyces coelicolor M1152. This work represents the first cloning and characterization of an ICZ gene cluster isolated from a marine-derived actinomycete strain and would be helpful for thoroughly understanding the biosynthetic mechanism of ICZ glycosides.

  3. Redox Impact on Starch Biosynthetic Enzymes in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Skryhan, Katsiaryna

    Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism are coordina...... of these amino acids for targeted stress-tolerant enzyme bioengineering.......Summary The thesis provides new insight into the influence of the plant cell redox state on the transient starch metabolism in Arabidopsis thaliana with a focus on starch biosynthetic enzymes. Two main hypotheses forms the basis of this thesis: 1) photosynthesis and starch metabolism...... are coordinated by the redox state of the cell via post-translational modification of the starch metabolic enzymes containing redox active cysteine residues and these cysteine residues became cross-linked upon oxidation providing a conformational change leading to activity loss; 2) cysteine residues...

  4. 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.

  5. Pictet–Spengler reaction-based biosynthetic machinery in fungi

    OpenAIRE

    Yan, Wei; Ge, Hui Ming; Wang, Gang; Jiang, Nan; Mei, Ya Ning; Jiang, Rong; Li, Sui Jun; Chen, Chao Jun; Jiao, Rui Hua; Xu, Qiang; Ng, Seik Weng; Tan, Ren Xiang

    2014-01-01

    The Pictet–Spengler (PS) reaction constructs many important phytochemicals such as morphine and camptothecin, but it has not yet been noticed in the fungal kingdom. Here, the startup of the PS reaction-based silent fungal biosynthetic machinery is presented to generate unforeseeably “unnatural” natural products of unprecedented carbon skeletons with antibacterial and acetylcholinesterase inhibitory activities. The gene-implied enzyme inhibition strategy is introduced to facilitate understandi...

  6. Enhancement of epidermal regeneration by biosynthetic epidermal growth factor

    OpenAIRE

    1986-01-01

    Epidermal regeneration depends on mitosis and migration of keratinocytes. Epidermal growth factor is known to stimulate growth of keratinocytes in vitro, thus it might be expected to promote wound healing. The results of this study show that topical application of biosynthetic human epidermal growth factor accelerates epidermal regeneration in split-thickness wounds and partial-thickness burns. The significant enhancement of epidermal regeneration suggests the potential for clinical use of ep...

  7. The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

    OpenAIRE

    Lawrence, J. G.; Roth, J R

    1995-01-01

    The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase...

  8. Biosynthetic Analysis of the Petrobactin Siderophore Pathway from Bacillus anthracis▿

    OpenAIRE

    Lee, Jung Yeop; Janes, Brian K.; Passalacqua, Karla D; Pfleger, Brian F.; Bergman, Nicholas H; Liu, Haichuan; Håkansson, Kristina; Somu, Ravindranadh V.; Aldrich, Courtney C.; Cendrowski, Stephen; Hanna, Philip C.; Sherman, David H.

    2006-01-01

    The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of...

  9. THE CAROTENOID BIOSYNTHETIC PATHWAY: THINKING IN ALL DIMENSIONS

    OpenAIRE

    Shumskaya, Maria; Wurtzel, Eleanore T.

    2013-01-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signalling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavour of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and...

  10. New method for isolating barophiles from intestinal contents of deep-sea fishes retrieved from the abyssal zone.

    Science.gov (United States)

    Nakayama, A; Yano, Y; Yoshida, K

    1994-11-01

    We devised a new method (the dorayaki method) using marine agar under in situ pressures to isolate barophilic bacteria from the intestinal contents of three deep-sea fishes (two Coryphaenoides yaquinae samples and one Ilyophis sp. sample) retrieved from depths of 4,700 to 6,100 m in the Northwest Pacific Ocean. All 10 strains isolated from one sample (C. yaquinae) were obligately barophilic. One of the 10 strains did not grow at atmospheric pressure and 103.4 MPa but did grow well between 20.7 and 82.7 MPa, with optimal growth at 41.4 MPa. This method is useful for isolating psychrophilic and barophilic deep-sea bacteria. PMID:16349450

  11. Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: A comparative study of two methods

    Directory of Open Access Journals (Sweden)

    Prasad S

    2009-04-01

    Full Text Available Detection of slime in Pseudomonas aeruginosa can be useful in understanding the virulence of this organism. Here, comparative studies of two phenotypic methods using the tube method and the spectrophotometric method for slime production from 100 clinically and 21 environmentally significant isolates of P. aeruginosa were performed. A total of 68 isolates were positive by either of the tests whereas only 34 were positive by both the tests. The tube method detected slime significantly in more number of isolates than the spectrophotometric method. The tube test was found to be superior to the spectrophotometric method in ease of performance, interpretation and sensitivity. Among the clinical isolates, systemic isolates produce less slime compared to wound, respiratory and urinary isolates. Isolates from the hospital environment produced more slime indicating that this virulence marker helps the organism to survive for longer periods and cause nosocomial infections.

  12. Evaluation of the Etest Method for Determining Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media

    OpenAIRE

    Pfaller, M A; Messer, S. A.; Karlsson, Å.; Bolmström, A.

    1998-01-01

    The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35°C. The yeast isolates...

  13. Evaluation of Etest Method for Determining Caspofungin (MK-0991) Susceptibilities of 726 Clinical Isolates of Candida Species

    OpenAIRE

    Pfaller, M A; Messer, S. A.; Mills, K.; Bolmström, A.; Jones, R N

    2001-01-01

    The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 isolates of Candida spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. MICs were determined by Etest for all 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35°C. The Candida isola...

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

    International Nuclear Information System (INIS)

    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)

  15. 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 ...

  16. Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene.

    OpenAIRE

    Sá-Correia, I.; Darzins, A; Wang, S K; Berry, A.; Chakrabarty, A M

    1987-01-01

    The specific activities of phosphomannose isomerase (PMI), phosphomannomutase (PMM), GDP-mannose pyrophosphorylase (GMP), and GDP-mannose dehydrogenase (GMD) were compared in a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa and in two spontaneous nonmucoid revertants. In both revertants some or all of the alginate biosynthetic enzymes we examined appeared to be repressed, indicating that the loss of the mucoid phenotype may be a result of decreased formation of sugar-nucleotide prec...

  17. Development of a System and Method for Automated Isolation of Stromal Vascular Fraction from Adipose Tissue Lipoaspirate

    Directory of Open Access Journals (Sweden)

    Swathi SundarRaj

    2015-01-01

    Full Text Available Autologous fat grafting for soft tissue reconstruction is challenged by unpredictable long-term graft survival. Fat derived stromal vascular fraction (SVF is gaining popularity in tissue reconstruction as SVF-enriched fat grafts demonstrate improved engraftment. SVF also has potential in regenerative medicine for remodeling of ischemic tissues by promoting angiogenesis. Since SVF cells do not require culture expansion, attempts are being made to develop automated devices to isolate SVF at the point of care. We report development of a closed, automated system to process up to 500 mL lipoaspirate using cell size-dependent filtration technology. The yield of SVF obtained by automated tissue digestion and filtration (1.17 ± 0.5 × 105 cells/gram was equivalent to that obtained by manual isolation (1.15 ± 0.3 × 105; p = 0.8, and the viability of the cells isolated by both methods was greater than 90%. Cell composition included CD34+CD31− adipose stromal cells, CD34+CD31+ endothelial progenitor cells, and CD34−CD31+ endothelial cells, and their relative percentages were equivalent to SVF isolated by the manual method. CFU-F capacity and expression of angiogenic factors were also comparable with the manual method, establishing proof-of-concept for fully automated SVF isolation, suitable for use in reconstructive surgeries and regenerative medicine applications.

  18. Ground Anthrax Bacillus Refined Isolation (GABRI) method for analyzing environmental samples with low levels of Bacillus anthracis contamination

    OpenAIRE

    Fasanella, Antonio; Di Taranto, Pietro; Garofolo, Giuliano; Colao, Valeriana; Marino, Leonardo; Buonavoglia, Domenico; Pedarra, Carmine; Adone, Rosanna; Hugh-Jones, Martin

    2013-01-01

    Background In this work are reported the results of a qualitative analytical method capable of detecting Bacillus anthracis spores when they are present in very low concentration in the soil. The Ground Anthrax Bacillus Refined Isolation (GABRI) method, assessed in our laboratory, was compared with the classic method. The comparison involved artificially anthrax-contaminated soil samples (500 spores/7.5 grams soil) and naturally contaminated soil samples collected in Bangladesh during a field...

  19. Collaborative study of a method for the isolation of light filth from breading of frozen food products.

    Science.gov (United States)

    Kvenberg, J E

    1975-05-01

    A method has been developed for the isolation of light filth from food breadings. The method involves a detergent boil, wet sieving, and flotation in an acid-alcohol, mineral oil flotation system in a Corning percolator. Collaborative studies resulted in clean filter papers and acceptable recoveries of added rodent hairs and insect fragments. The method has been adopted as official first action. PMID:1141169

  20. Insights into the evolution of macrolactam biosynthesis through cloning and comparative analysis of the biosynthetic gene cluster for a novel macrocyclic lactam, ML-449.

    Science.gov (United States)

    Jørgensen, Hanne; Degnes, Kristin F; Dikiy, Alexander; Fjaervik, Espen; Klinkenberg, Geir; Zotchev, Sergey B

    2010-01-01

    A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the beta-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis. PMID:19854930

  1. Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449 ▿ †

    Science.gov (United States)

    Jørgensen, Hanne; Degnes, Kristin F.; Dikiy, Alexander; Fjærvik, Espen; Klinkenberg, Geir; Zotchev, Sergey B.

    2010-01-01

    A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the β-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis. PMID:19854930

  2. 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. PMID:27072286

  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. A rapid method of fruit cell isolation for cell size and shape measurements

    Directory of Open Access Journals (Sweden)

    Johnston Jason W

    2009-04-01

    tissue from all these fruit types, showing a broad utility to this protocol. Conclusion We have developed a method for isolating single cells from fleshy fruit that reduces the time needed for fruit cell separation. This method was used to demonstrate differences in cell size and shape for 5 different apple cultivars. While firmness between the different cultivars is independent of cell size, apples with more angular cells appear to be firmer.

  5. An easy, rapid method to isolate RPE cell protein from the mouse eye.

    Science.gov (United States)

    Wei, Hong; Xun, Zixian; Granado, Herta; Wu, Angela; Handa, James T

    2016-04-01

    The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination. PMID:26424220

  6. A SIMPLE METHOD FOR ISOLATION OF SOME BACILLUS STRAINS WITH AN EXPRESSED ANTI-CANCER ACTIVITY

    Directory of Open Access Journals (Sweden)

    Francesco Marotta

    2006-02-01

    Full Text Available ABSTRACT:There is now increasing evidence that probiotic bacteria can provide health benefits to humans. In many areas of medicine (gastroenterology, urology, allergology, oncology and others, these sanative microorganisms may be considered as possible and viable alternatives applicable to patient care. Particularly, we have found that oral administration of Bacillus oligonitrophilus KU-1 cells can be used for treatment and prevention of some tumors. Here we present a simple method for isolation of bacteria with anticancer properties from soil.RESUMEN:Está aumentando la evidencia de que hay bacterias probióticas que pueden proporcionar beneficios saludables a los seres humanos. En muchas áreas de la medicina (gastroenterología, urología, alergología, oncología y otras, estos microorganismos pueden considerarse como alternativas posibles y viables aplicables al cuidado del paciente. Particularmente, nosotros hemos encontrado que la administración oral de células KU-1 Bacillus oligonitrophilus puede ser utilizada para el tratamiento y la prevención de algunos tumores. Aquí presentamos un método simple para aislamiento de suelos, de bacterias con características anticáncer.

  7. Radioactive beams produced by the ISOL method: development for laser ionization and for surface ionization

    International Nuclear Information System (INIS)

    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)

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

    International Nuclear Information System (INIS)

    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)

  9. A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.

    Science.gov (United States)

    Kaelberer, Melanie Maya; Jordt, Sven-Eric

    2016-01-01

    Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined. PMID:27168016

  10. Evaluation of ERIC-PCR as Genotyping Method for Corynebacterium pseudotuberculosis Isolates

    Science.gov (United States)

    Dorneles, Elaine M. S.; Santana, Jordana A.; Ribeiro, Dayana; Dorella, Fernanda Alves; Guimarães, Alessandro S.; Moawad, Mohamed S.; Selim, Salah A.; Garaldi, Ana Luiza M.; Miyoshi, Anderson; Ribeiro, Márcio G.; Gouveia, Aurora M. G.; Azevedo, Vasco; Heinemann, Marcos B.; Lage, Andrey P.

    2014-01-01

    The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations. PMID:24901343

  11. A simple method for isolation of Gypsophila saponins for the combined application of targeted toxins and saponins in tumor therapy.

    Science.gov (United States)

    Weng, Alexander; Bachran, Diana; Görick, Cornelia; Bachran, Christopher; Fuchs, Hendrik; Melzig, Matthias F

    2009-10-01

    Saponinum album (SAP) is a complex mixture of triterpene saponins from Gypsophila paniculata L. Although most of the saponins from SAP are characterized, the separation of pure saponins remains time consuming and costly, involving different chromatographic techniques. Recently it was shown that SAP drastically enhanced the cytotoxicity of a chimeric toxin consisting of the N-glycosidase saporin and human epidermal growth factor (Sap-EGF) in cell culture experiments. In view of a potential therapeutic use of the coadministration of SAP and Sap-EGF in tumor therapy, an economic and time-saving method for the isolation of pure saponins from the crude SAP mixture in high amounts is required. In this study we isolated a single saponin by a simple chromatographic method. The isolated saponin was characterized by mass spectrometry and was shown to enhance the cytotoxicity of Sap-EGF on HER14 cells. PMID:19452437

  12. STUDIES ON DRUG-RESISTANCE PATTERN BY PHENOTYPIC METHODS IN Mycobacterium tuberculosis ISOLATES IN A TERTIARY CARE HOSPITAL Authors

    Directory of Open Access Journals (Sweden)

    PATIL S.D.

    2013-10-01

    Full Text Available Background: Tuberculosis is a major health problem in India. The situation has been made worse because of the emergence of drug resistance. Tuberculosis has affected more than one third of the world’s population and India contributes to ¼ of the global annual incidence. With ¼ of the drug resistant forms coming from India. With this context the present study was undertaken to assess the magnitude and pattern of drug resistance among the isolates of Mycobacterium tuberculosis.Aims & Objective: To detect the drug-resistance pattern of the isolates of Mycobacterium tuberculosis from a tertiary care hospital. The objective was achieved by screening of sputum smears for acid-fast bacilli ( AFB, culture of AFB positive samples on Lowenstein-Jensen’s (L.J., identification of isolates of Mycobacterium tuberculosis and drug susceptibility testing (DST against first-line and second-line anti-tuberculosis drugs.Methods: Standard procedures were followed for the isolation & identification of Mycobacterium tuberculosis. DST was carried out by proportion method on L.J. to detect drug resistance pattern.Results: Out of 1186 samples studied, 123 were AFB & culture positive strains of Mycobacterium tuberculosis. Of these 123 strains 10 isolates were Multi-drug resistant (MDR-TB showing resistance to both Isoiniazide and Rifampicin. Out of these 10 strains two are Extensively drug-resistant (XDR-TB strains -one is resistant to kanamycin, ethionamide, ciprofloxacin and rifabutin while the other is resistant to kanamycin, D-Cycloserine, ciprofloxacin and rifabutin. Mono-drug resistance to D-cycloserine was observed in one isolate while resistance to two drugs like ethionamide, P-amino salicylic acid; ethionamide, ciprofloxacin and ethionamide, D-Cycloserine was observed in three isolates.Conclusion: The present work helps to study & monitor antibiotic susceptibility test results. Moreover its documentation will make us aware of any change in

  13. The height-width ratio limited value for rubber bearing isolated structure computed by uniform design method

    Institute of Scientific and Technical Information of China (English)

    WANG Tie-ying; WANG Huan-ding; ZHANG Yong-shan; LIU Wen-guang

    2007-01-01

    Rubber isolation is the most mature control technology in practical application, and is widely used by short rigid buildings. However, many high isolation buildings have been built around the world in recent years,which do not follow the existing criterions and codes. Many researchers began to research the special problems caused by larger height-width ratio isolation structures. The overturning effect of high height-width ratio structures with rubber bearing is firstly studied. Considering the main factors, such as the height-width ratio of structures, type of site, the designed basic acceleration of ground motion and the decouple factor in horizon, computing experiment is defined with the Uniform Design Method, which is also known as designing isolation structure. The forces of the bearing under edge of structures based on the position of the rubber bearing are calculated. The result indicates that the rubber bearings will lose its functionality under very high tension and compressing force of earthquake motion in horizon and vertical, when the height-width ratio is over a certain value.Thus, based on the calculation result of isolation structures defined in the uniform design method, regression analysis is conducted, and also the rubber edge force regression formula are gotten, which has higher correlation and smaller standard deviation. This formula can be used to roughly calculate whether the pull force occurs at the edge of the building. By the edge bearings of isolation structure minimum force formula, the height-width ratio limited value of the isolation structure is deducted when rubber bearing has minimum force of zero.

  14. Isolation of Tricin, Luteolin, and Quercetin Flavonoids from Syrian Artemisia Vulgaris L., and Determination Their Structure By Spectroscopic Methods

    International Nuclear Information System (INIS)

    The crude acetonic extract of Syrian Artemisia vulgaris L. was fractionated by chromatographic methods and yielded three known flavonoids, Tricin, Luteolin, Quercetin. UV, IR, Mass spectroscopy, and 1D and 2D NMR techniques were used to determine the structure of isolated compounds.(author)

  15. 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. PMID:25938329

  16. Evaluation and optimization of avian embryos and cell culture methods for efficient isolation and propagation of avian influenza viruses

    Science.gov (United States)

    Surveillance of wild bird populations for avian influenza viruses (AIV) contributes to our understanding of AIV evolution and ecology. Both real-time reverse transcriptase polymerase chain reaction (RRT-PCR) and virus isolation in embryonating chicken eggs (ECE) are standard methods for detecting A...

  17. A rapid RT-PCR based method to isolate complementary DNA fragments flanking retrovirus integration sites.

    OpenAIRE

    1997-01-01

    Proto-oncogenes in retrovirally induced myeloid mouse leukemias are frequently activated following retroviral insertion. The identification of common virus integration sites (VISs) and isolation of the transforming oncogene is laborious and time consuming. We established a rapid and simple PCR based procedure which facilitates the identification of VISs and novel proto-oncogenes. Complementary DNA fragments adjacent to retrovirus integration sites were selectively isolated by applying a rever...

  18. Alterations in the heme biosynthetic pathway as an index of exposure to toxins

    Energy Technology Data Exchange (ETDEWEB)

    Marks, G.S.; Zelt, D.T.; Cole, S.P.

    1982-07-01

    Under normal circumstances the heme biosynthetic pathway is carefully controlled and porphyrins are formed in only trace amounts. When control mechanisms are disturbed by xenobiotics, porphyrins may be formed and serve as a signal of the interaction between a xenobiotic and the heme biosynthetic pathway. For example, porphyrinuria was an early manifestation of a hexachlorobenzene-induced porphyria outbreak in Turkey. In humans exposed to polybrominated biphenyls and to 2,3,7,8-tetrachlorodibenzo-p-dioxin the urinary porphyrin pattern was significantly different from normal in a large number of exposed individuals. The question is raised whether measurement of urinary porphyrin profiles by improved methods will enable an estimate to be made of the extent of exposure to haloaromatic hydrocarbons in the human population. A wide variety of xenobiotics interact with the prosthetic heme of cytochrome P-450 forming novel N-alkylporphyrins. Identification of these N-alkylporphyrins in body fluids might provide a means of assessing exposure to a variety of xenobiotics in human populations.

  19. Stress and developmental responses of terpenoid biosynthetic genes in Cistus creticus subsp. creticus.

    Science.gov (United States)

    Pateraki, Irene; Kanellis, Angelos K

    2010-06-01

    Plants, and specially species adapted in non-friendly environments, produce secondary metabolites that help them to cope with biotic or abiotic stresses. These metabolites could be of great pharmaceutical interest because several of those show cytotoxic, antibacterial or antioxidant activities. Leaves' trichomes of Cistus creticus ssp. creticus, a Mediterranean xerophytic shrub, excrete a resin rich in several labdane-type diterpenes with verified in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. Bearing in mind the properties and possible future exploitation of these natural products, it seemed interesting to study their biosynthesis and its regulation, initially at the molecular level. For this purpose, genes encoding enzymes participating in the early steps of the terpenoids biosynthetic pathways were isolated and their gene expression patterns were investigated in different organs and in response to various stresses and defence signals. The genes studied were the CcHMGR from the mevalonate pathway, CcDXS and CcDXR from the methylerythritol 4-phosphate pathway and the two geranylgeranyl diphosphate synthases (CcGGDPS1 and 2) previously characterized from this species. The present work indicates that the leaf trichomes are very active biosynthetically as far as it concerns terpenoids biosynthesis, and the terpenoid production from this tissue seems to be transcriptionally regulated. Moreover, the CcHMGR and CcDXS genes (the rate-limiting steps of the isoprenoids' pathways) showed an increase during mechanical wounding and application of defence signals (like meJA and SA), which is possible to reflect an increased need of the plant tissues for the corresponding metabolites. PMID:20364257

  20. A rapid method for isolation of low-molecular-weight RNA from Arabidopsis using low salt concentration buffer

    Directory of Open Access Journals (Sweden)

    Han Cheng

    2010-08-01

    Full Text Available Normal 0 7.8 pt 0 2 false false false EN-US ZH-CN X-NONE MicrosoftInternetExplorer4 We have developed a rapid extraction method using low salt concentration buffer for the isolation of low-molecular-weight RNA from Arabidopsis tissues. The method was quick and efficient, and the small scale extraction process took no more than 1 hour, while yield and RNA quality were comparable with those of previously reported. The LMW RNA isolated using this method was high quality, abundant in small RNA and free of high molecular weight RNA. This method can be used to extract low-molecular-weight RNA for the purpose of small RNA cloning and detection, and library construction.

  1. Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Ziyu; Lasure, Linda L; Magnuson, Jon K

    2014-05-27

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  2. A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry.

    OpenAIRE

    Gergely, L.; Cook, L.; Agnello, V

    1997-01-01

    A method for Ca2+ flux measurement on isolated human peripheral B cells that uses flow cytometry is described. B cells were isolated by anti-CD19 magnetic bead sorting, and Ca2+ flux was measured with the fluo-3 reagent on a standard single-laser flow cytometer. The response of B-cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protein A, concanavalin A, and ionomycin was determined. Percentage of responder B cells, the level of Ca2+, and the time of peak stimulation were measu...

  3. 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)

    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

  4. Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus

    Science.gov (United States)

    Dai, Ziyu; Lasure, Linda L.; Magnuson, Jon K.

    2008-11-11

    The present invention encompasses isolated gene regulatory elements and gene transcription terminators that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention also encompasses a method of utilizing a fungus for protein or chemical production. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to another molecule comprising a coding region of a gene of interest. The gene regulatory element and gene transcription terminator may temporally and spatially regulate expression of particular genes for optimum production of compounds of interest in a transgenic fungus.

  5. Comparison of ultracentrifugation and density gradient separation methods for isolating Tca8113 human tongue cancer cell line-derived exosomes

    OpenAIRE

    Zhang, Zhuoyuan; Wang, Chenxing; Li, Tang; LIU, ZHE; LI, LONGJIANG

    2014-01-01

    The aim of the present study was to compare the method of ultracentrifugation and density gradient separation for isolating Tca8113 human tongue squamous cell carcinoma cell line-derived exosomes. The exosomes were obtained from the culture supernatant of cultured Tca8113 cells, respectively, followed by identification with transmission electron microscopy observation and western blot analysis. The two different methods were then compared by the morphology, the distribution range of the parti...

  6. Experimental evaluation of sand fly collection and storage methods for the isolation and molecular detection of Phlebotomus-borne viruses

    OpenAIRE

    Remoli, Maria Elena; Bongiorno, Gioia; Fortuna, Claudia; Marchi, Antonella; Bianchi, Riccardo; Khoury, Cristina; Ciufolini, Maria Grazia; Gramiccia, Marina

    2015-01-01

    Background Several viruses have been recently isolated from Mediterranean phlebotomine sand flies; some are known to cause human disease while some are new to science. To monitor the Phlebotomus-borne viruses spreading, field studies are in progress using different sand fly collection and storage methods. Two main sampling techniques consist of CDC light traps, an attraction method allowing collection of live insects in which the virus is presumed to be fairly preserved, and sticky traps, an ...

  7. Extraction of Saponin from Camellia oleifera Abel Cake by a Combination Method of Alkali Solution and Acid Isolation

    OpenAIRE

    Yongjun Liu; Zhifeng Li; Hongbo Xu; Yuanyuan Han

    2016-01-01

    Saponin 15%~20% content in the seed cake of Camellia oleifera Abel, from which Camellia oil is squeezed, is a natural nonionic surface active agent and is extensively applied to emulsification, humectation, foaming, medicine, pesticide, and so on. In this paper, the extraction process of saponin was researched through a combining method of alkali solution and acid isolation. A quantitative method for saponin was established by ultraviolet spectrophotometer. The influence of extraction factors...

  8. Limited collaborative study of an optional method for the isolation of light filth from fig and fruit paste.

    Science.gov (United States)

    Kvenberg, J E; Eisenberg, W V; King, A C

    1975-05-01

    The present official first action method for the isolation of light filth from fig and fruit paste, 44.083(a), occasionally yields excessive plant debris on filter papers, which causes difficulty in effectively counting insect filth. The proposed method provides the option of an additional flotation of trapped off material in a Corning percolator. This revision has been adopted as official first action as an alternative to 44.083(a). PMID:1141170

  9. Carotenoid biosynthetic genes in Brassica rapa: comparative genomic analysis, phylogenetic analysis, and expression profiling

    OpenAIRE

    Li, Peirong; Zhang, Shujiang; Zhang, Shifan; Li, Fei; Zhang, Hui; Cheng, Feng; Wu, Jian; Wang, Xiaowu; Sun, Rifei

    2015-01-01

    Background Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms. Despite much research on carotenoid biosynthesis in the model plant Arabidopsis thaliana, there is a lack of information on the carotenoid pathway in Brassica rapa. To better understand its carotenoid biosynthetic pathway, we performed a systematic analysis of carotenoid biosynthetic genes at the genome level in B. rapa. Results We identified 67 carotenoid biosynthetic genes in B. rapa, which were ort...

  10. Modified PAP method to detect heteroresistance to vancomycin among methicillin resistant Staphylococcus aureus isolates at a tertiary care hospital

    Directory of Open Access Journals (Sweden)

    Iyer R

    2008-01-01

    Full Text Available This study was an attempt at developing, establishing, validating and comparing the modified PAP method for detection of hetero-vancomycin resistant Staphylococcus aureus (h-VRSA with the routine antimicrobial susceptibility testing (using the BSAC standardized disc diffusion method, minimum inhibitory concentrations of vancomycin using standard E-test methodology and the Hiramatsu′s screening method. A total of 50 methicillin resistant Staphylococcus aureus obtained from various clinical specimens, along with the Mu 3 and Mu 50 strains as controls, were studied. No VRSA isolates were obtained. However, four of the test strains were positive by the Hiramatsu′s screening method, of which only one isolate could be confirmed by the modified PAP analysis method. This isolate was a coloniser from the drain fluid of a liver transplant recipient. The sensitivity, specificity, positive predictive value and the overall efficiency of the Hiramatsu′s screening method with the modified PAP analysis as the gold standard were found to be 100, 93.8, 25 and 94%, respectively. It is very essential for clinical laboratories to screen for h-VRSA, given the increasing use of glycopeptide antibiotics in therapy and the potential for failed therapy in patients infected with these strains.

  11. 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.

  12. A Fault Detection and Isolation Scheme Based on Parity Space Method for Discrete Time-delay System

    Institute of Scientific and Technical Information of China (English)

    WANG Hong-yu; TIAN Zuo-hua; SHI Song-jiao; WENG Zheng-xin

    2008-01-01

    A Fault detection and isolation (FDI) scheme for discrete time-delay system is proposed in this paper, which can not only detect but also isolate the faults. A time delay operator ▽ is introduced to resolve the problem brought by the time-delay system. The design and computation for the FDI system is carried by computer math tool Maple, which can easily deal with the symbolic computation. Residuals in the form of parity space can be deduced from the recursion of the system equations. Further mote, a generalized residual set is created using the freedom of the parity space redundancy. Thus, both fault detection and fault isolation have been accomplished. The proposed method has been verified by a numerical example.

  13. 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 that...

  14. Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

    OpenAIRE

    T.H.T Pham

    2013-01-01

      The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random coil-like connector, and B is a second self-assembling block. A and B have to be chosen such that they do not cross-assemble. With these molecules it should be possible to fabricate hydrogels in whi...

  15. Survey of volatile oxylipins and their biosynthetic precursors in bryophytes.

    Science.gov (United States)

    Croisier, Emmanuel; Rempt, Martin; Pohnert, Georg

    2010-04-01

    Oxylipins are metabolites which are derived from the oxidative fragmentation of polyunsaturated fatty acids. These metabolites play central roles in plant hormonal regulation and defense. Here we survey the production of volatile oxylipins in bryophytes and report the production of a high structural variety of C5, C6, C8 and C9 volatiles of mosses. In liverworts and hornworts oxylipin production was not as pronounced as in the 23 screened mosses. A biosynthetic investigation revealed that both, C18 and C20 fatty acids serve as precursors for the volatile oxylipins that are mainly produced after mechanical wounding of the green tissue of mosses. PMID:20079505

  16. Evaluation of a Wet Chemistry Method for Isolation of Cyclotron Produced [211At]Astatine

    Directory of Open Access Journals (Sweden)

    Shigeki Watanabe

    2013-09-01

    Full Text Available A “wet chemistry” approach for isolation of 211At from an irradiated bismuth target is described. The approach involves five steps: (1 dissolution of bismuth target in conc. HNO3; (2 removal of the HNO3 by distillation; (3 dissolution of residue in 8 M HCl; (4 extraction of 211At from 8 M HCl into DIPE; and (5 extraction of 211At from DIPE into NaOH. Results from 55 “optimized” 211At isolation runs gave recovery yields of approximately 78% after decay and attenuation corrections. An attenuation-corrected average of 26 ± 3 mCi in the target provided isolated (actual yields of 16 ± 3 mCi of 211At. A sixth step, used for purification of 211At from trace metals, was evaluated in seven runs. In those runs, isolated 211At was distilled under reductive conditions to provide an average 71 ± 8% recovery. RadioHPLC analyses of the isolated 211At solutions, both initial and after distillation, were obtained to examine the 211At species present. The primary species of 211At present was astatide, but astatate and unidentified species were also observed. Studies to determine the effect of bismuth attenuation on 211At were conducted to estimate an attenuation factor (~1.33 for adjustment of 211At readings in the bismuth target.

  17. 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

  18. Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

    Science.gov (United States)

    Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

    2016-01-01

    The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome. PMID:27586421

  19. Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.

    NARCIS (Netherlands)

    Rood, I.M.; Deegens, J.K.J.; Merchant, M.L.; Tamboer, W.P.M.; Wilkey, D.W.; Wetzels, J.F.M.; Klein, J.B.

    2010-01-01

    Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficie

  20. 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

  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)

    Rulings in 2001 and 2006 by the United States Supreme Court concerning the protection of Geographically Isolated Wetlands (GIWs) unveiled a critical area of research: quantifying the extent of potential hydrologic connectivity of GIWs to navigable waters and their effects at a va...

  3. Substrate specificity of the sialic acid biosynthetic pathway

    Energy Technology Data Exchange (ETDEWEB)

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  4. Structural Insights Into the Evolutionary Paths of Oxylipin Biosynthetic Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, D.-S.; Nioche, P.; Hamberg, M.; Raman, C.S.

    2009-05-20

    The oxylipin pathway generates not only prostaglandin-like jasmonates but also green leaf volatiles (GLVs), which confer characteristic aromas to fruits and vegetables. Although allene oxide synthase (AOS) and hydroperoxide lyase are atypical cytochrome P450 family members involved in the synthesis of jasmonates and GLVs, respectively, it is unknown how these enzymes rearrange their hydroperoxide substrates into different products. Here we present the crystal structures of Arabidopsis thaliana AOS, free and in complex with substrate or intermediate analogues. The structures reveal an unusual active site poised to control the reactivity of an epoxyallylic radical and its cation by means of interactions with an aromatic {pi}-system. Replacing the amino acid involved in these steps by a non-polar residue markedly reduces AOS activity and, unexpectedly, is both necessary and sufficient for converting AOS into a GLV biosynthetic enzyme. Furthermore, by combining our structural data with bioinformatic and biochemical analyses, we have discovered previously unknown hydroperoxide lyase in plant growth-promoting rhizobacteria, AOS in coral, and epoxyalcohol synthase in amphioxus. These results indicate that oxylipin biosynthetic genes were present in the last common ancestor of plants and animals, but were subsequently lost in all metazoan lineages except Placozoa, Cnidaria and Cephalochordata.

  5. The carotenoid biosynthetic pathway: thinking in all dimensions.

    Science.gov (United States)

    Shumskaya, Maria; Wurtzel, Eleanore T

    2013-07-01

    The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signaling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavor of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and predictability of metabolic engineering of carotenoids rests in the ability to target carotenoids for specific physiological purposes as well as to simultaneously modify carotenoids along with other desired traits. Here, we ask whether predictive metabolic engineering of the carotenoid pathway is indeed possible. Despite a long history of research on the pathway, at this point in time we can only describe the pathway as a parts list and have almost no knowledge of the location of the complete pathway, how it is assembled, and whether there exists any trafficking of the enzymes or the carotenoids themselves. We discuss the current state of knowledge regarding the "complete" pathway and make the argument that predictive metabolic engineering of the carotenoid pathway (and other pathways) will require investigation of the three dimensional state of the pathway as it may exist in plastids of different ultrastructures. Along with this message we point out the need to develop new types of visualization tools and resources that better reflect the dynamic nature of biosynthetic pathways. PMID:23683930

  6. Characterization of dominant lactic acid bacteria isolated from São Jorge cheese, using biochemical and ribotyping methods

    OpenAIRE

    Kongo, J.M.; Kongo, A. J.; Malcata, F. X.; Wiedman, M.

    2007-01-01

    Aims: To identify, using phenotypic and genotypic methods, the dominant lactic acid bacteria (LAB) present in São Jorge cheese – one of the 11 Portuguese cheeses currently bearing an Appéllation d’Origine Protegée status. Methods and Results:  A total of 225 isolates from milk, curd and cheeses throughout ripening were identified to the genus level, 108 to the species level and ten to the strain level. Phenotypic methods indicated that lactobacilli, followed by enterococci, were the domina...

  7. 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

    This paper proposes the Waveform Reconstruction Method (WRM), which is utilized in the single-phase Series Active Filter's (SAF's) control algorithm, in order to extract the load harmonic voltage component of voltage harmonic type single-phase diode rectifier loads. Employing WRM and the line...... 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.......5 kW single-phase SAF compensated system prove the theory....

  8. Extraction of Saponin from Camellia oleifera Abel Cake by a Combination Method of Alkali Solution and Acid Isolation

    Directory of Open Access Journals (Sweden)

    Yongjun Liu

    2016-01-01

    Full Text Available Saponin 15%~20% content in the seed cake of Camellia oleifera Abel, from which Camellia oil is squeezed, is a natural nonionic surface active agent and is extensively applied to emulsification, humectation, foaming, medicine, pesticide, and so on. In this paper, the extraction process of saponin was researched through a combining method of alkali solution and acid isolation. A quantitative method for saponin was established by ultraviolet spectrophotometer. The influence of extraction factors was investigated by a single-factor test and a response surface methodology. The results indicated that the optimal extraction conditions of saponin were extraction temperature 68°C, alkali solution pH 9.1, acid isolation pH 4.1, and liquid-solid ratio 15.9 : 1. The extraction rate of saponin was 76.12% at the optimal extraction conditions.

  9. Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

    Science.gov (United States)

    Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa; Hirai, Itaru

    2015-06-01

    Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. PMID:25809972

  10. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis

    Directory of Open Access Journals (Sweden)

    Poliana de Castro Melo

    2013-01-01

    Full Text Available Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well "U" bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production.

  11. Comparison of Phenotypical and Molecular Methods for the Identification of Bacterial Strains Isolated from a Deep Subsurface Environment

    OpenAIRE

    Boivin-Jahns, V.; Bianchi, A.; Ruimy, R; Garcin, J.; Daumas, S.; Christen, R

    1995-01-01

    Seventy-four bacterial strains were freshly isolated from a mine gallery. Using these bacteria, we have investigated how a molecular identification based on the analysis of small subunit rDNA sequences would compare in terms of precision and reliability to a more classical comparison of phenotypical descriptions (100 morphological and physiological traits). Our data clearly showed that a phylogenetic analysis of small subunit rDNA sequences is more efficient than classical phenotypic methods ...

  12. Post-irradiation viability and cytotoxicity of natural killer cells isolated from human peripheral blood using different methods.

    Science.gov (United States)

    Hietanen, Tenho; Pitkänen, Maunu; Kapanen, Mika; Kellokumpu-Lehtinen, Pirkko-Liisa

    2016-01-01

    Purpose We compared the pre- and post-irradiation viability and cytotoxicity of human peripheral natural killer cell (NK) populations obtained using different isolation methods. Material and methods Three methods were used to enrich total NK cells from buffy coats: (I) a Ficoll-Paque gradient, plastic adherence and a nylon wool column; (II) a discontinuous Percoll gradient; or (III) the Dynal NK cell isolation kit. Subsequently, CD16(+) and CD56(+) NK cell subsets were collected using (IV) flow cytometry or (V) magnetic-activated cell sorting (MACS) NK cell isolation kits. The yield, viability, purity and cytotoxicity of the NK cell populations were measured using trypan blue exclusion, flow cytometry using propidium iodide and (51)Cr release assays after enrichments as well as viability and cytotoxicity after a single radiation dose. Results The purity of the preparations, as measured by the CD16(+) and CD56(+) cell content, was equally good between methods I-III (p = 0.323), but the content of CD16(+) and CD56(+) cells using these methods was significantly lower than that using methods IV and V (p = 0.005). The viability of the cell population enriched via flow cytometry (85.5%) was significantly lower than that enriched via other methods (99.4-98.0%, p = 0.003). The cytotoxicity of NK cells enriched using methods I-III was significantly higher than that of NK cells enriched using methods IV and V (p = 0.000). In vitro the NK cells did not recover cytotoxic activity following irradiation. In addition, we detected considerable inter-individual variation in yield, cytotoxicity and radiation sensitivity between the NK cells collected from different human donors. Conclusions The selection of the appropriate NK cell enrichment method is very important for NK cell irradiation studies. According to our results, the Dynal and MACS NK isolation kits best retained the killing capacity and the viability of irradiated NK cells. PMID:26634866

  13. Dendritic membrane from insect olfactory hairs: isolation method and electron microscopic observations.

    Science.gov (United States)

    Klein, U; Keil, T A

    1984-12-01

    Sensory hairs from antennae of male saturniid moths (Antheraea polyphemus) were separated while deep-frozen by shaking antennal branches with glass beads. The hairs were collected through their differential adhesion to the surface of a petri dish. The yield, determined by the length of the isolated hair fragments, was about 38% of the estimated total hair length per antenna. The dendritic membrane was separated from the hair fragments by centrifugation through Sephadex and further purified by ultracentrifugation in sucrose buffers. Transmission electron microscopy was used to monitor the steps of the hair and membrane isolation and to investigate the membrane pellet. Some membrane vesicles bound cationized ferritin, thus indicating a negatively charged cell surface coat. Negatively stained membrane vesicles exhibited a pattern of repetitive substructures irregularly distributed over the vesicle surface. The units had a diameter of about 3 nm and a maximal density of 30,000/micron2. PMID:6532523

  14. New Method for isolation of immunologically pure pili from Escherichia coli.

    OpenAIRE

    Korhonen, T K; Nurmiaho, E L; Ranta, H; Edén, C S

    1980-01-01

    A new technique for purification of bacterial pili was developed and applied to Escherichia coli strains isolated from the urine of patients with symptomatic urinary tract infections. After mechanical detachment from the bacterial cells, the pili were concentrated by precipitation with ammonium sulfate, dialyzed, and solubilized in buffer containing deoxycholate. The fraction containing the pili was purufied further by ultracentrifugation in a sucrose gradient and by elution through a Sepharo...

  15. A method of isoflavones isolation from red clover as standards for analyses

    OpenAIRE

    Piotr M. Górski; Stanisław Burda; Marian Jurzysta; Michał Płoszyński

    2013-01-01

    Five compounds having an isoflavone structure were isolated from the tops of red clover (Trifolium pratense). On the basis of spectral (UV, MS) and chromatographic (TLC, HPLC) analyses the compounds were identified as biochanin A, formononetin, pratensein, genistein and daidzein. Biochanin A and formononetin - two main clover estrogens - were obtained in crystalline forms in the amounts of 50 mg and 15 mg, respectively (per 250 g of D. W.). Homogenous fractions of pratensein, genistein, and d...

  16. Effect of Separation Method on Chemical Composition and Insecticidal Activity of Lamiaceae Isolates

    OpenAIRE

    Sajfrtová, M. (Marie); Sovová, H. (Helena); Karban, J. (Jindřich); Rochová, K. (Kristina); Pavela, R.; Barnet, M.

    2013-01-01

    Supercritical fluid extraction was used to isolate volatile compounds from savory (Satureja hortensis L.), thyme (Thymus vulgaris L.), lavender (Lavandula angustifolia L.) and peppermint (Mentha piperita L.). Three types of extracts were prepared using the benefit of variable solvent power of supercritical carbon dioxide under different extraction conditions. The composition and toxicity of CO2 extracts, products of Soxhlet extraction with hexane and ethanol, and essential oils obtained by h...

  17. Comparison of methods to differentiate staphylococcus and micrococcus species isolated from bovine mammary glands

    OpenAIRE

    Pengov A.; Jurčevič A.

    2003-01-01

    The hemolytic pattern of colonies, the slide coagulase (clumping factor) test, the tube coagulase test and the thermonuclease test were studied for their capability to differentiate Staphylococcus aureus from other Staphylococcus and Micrococcus species. A total 93 strains of Staphylococcus aureus isolated from bovine udder glands were tested. The hemolytic pattern of colonies and the slide coagulase test were found to be less reliable than other tests. No important differences could be found...

  18. Comparison of different methods for the isolation and purification of total community DNA from soil.

    Science.gov (United States)

    Krsek, M; Wellington, E M

    1999-12-01

    The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments. PMID:10579502

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

    Directory of Open Access Journals (Sweden)

    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].

  20. Nonlinear Biosynthetic Gene Cluster Dose Effect on Penicillin Production by Penicillium chrysogenum

    NARCIS (Netherlands)

    Nijland, Jeroen G.; Ebbendorf, Bjorg; Woszczynska, Marta; Boer, Remon; Bovenberg, Roel A. L.; Driessen, Arnold J. M.

    2010-01-01

    Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the beta-lactam biosynthetic p

  1. Stereoselective synthesis of deuterium-labeled (2S)-cyclohexenyl alanines, biosynthetic intermediates of cinnabaramide.

    Science.gov (United States)

    Barbie, Philipp; Huo, Liujie; Müller, Rolf; Kazmaier, Uli

    2012-12-01

    Dideuterated β-cyclohexenylalanines, proposed biosynthetic intermediates of the cinnabaramides, can be obtained from chiral alkynols via a sequence of Irland-Claisen rearrangement, ring closing metathesis, and radical decarboxylation. Feeding experiments indicate that both (2S)-β-cyclohexenylalanines can be incorporated into cinnabaramide, while the configuration at the cyclohexenyl ring does not restrict biosynthetic processing. PMID:23163839

  2. Variability in mycotoxin biosynthetic genes in Fusarium and its effect on mycotoxin contamination of crops

    Science.gov (United States)

    The Fusarium metabolites fumonisins and trichothecenes are among the mycotoxins of greatest concern to food and feed safety worldwide. As is the case for other fungal secondary metabolite biosynthetic genes, mycotoxin biosynthetic genes are often located adjacent to one another in gene clusters. Thu...

  3. Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

    NARCIS (Netherlands)

    Verdoes, J.C.; Sandmann, G.; Visser, H.; Diaz, M.; Mossel, van M.; Ooyen, van A.J.J.

    2003-01-01

    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both si

  4. Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples

    Science.gov (United States)

    Sundaram, Shivshankar; Prabhakarpandian, Balabhaskar; Pant, Kapil; Wang, Yi

    2014-01-01

    RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads

  5. Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction.

    Science.gov (United States)

    Tanriverdi, Kahraman; Kucukural, Alper; Mikhalev, Ekaterina; Tanriverdi, Selim E; Lee, Rosalind; Ambros, Victor R; Freedman, Jane E

    2016-05-15

    MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)-qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids. PMID:26969789

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

    Directory of Open Access Journals (Sweden)

    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.

  7. An alternative method to achieve one-lung ventilation by surgical pneumothorax in difficult lung isolation patient: a case report.

    Science.gov (United States)

    Yeh, Pin-Hung; Hsu, Po-Kai

    2016-04-01

    It is challenging to establish one-lung ventilation in difficult airway patients. Surgical pneumothorax under spontaneous breathing to obtain well-collapsed lung is a feasible method for thoracic surgery. A 76-year-old man with right empyema was scheduled for decortication. The patient had limited mouth opening due to facial cellulitis extending from the left cheek to neck. Generally, lung isolation is achieved by double-lumen endotracheal tube or bronchial blocker. Double-lumen tube insertion is difficult for patients with limited mouth opening and right-side placement of bronchial blocker usually causes insufficient deflation. We introduce an alternative lung isolation technique by surgical pneumothorax under spontaneous breathing simply with an endotracheal tube placement. This technique has never been applied into the management of difficult one-lung ventilation. By this method, we provide an ideal surgical condition with safer, less time-consuming, and less skill-demanding anesthesia. It would be an alternative choice for management of one-lung ventilation in the difficult lung isolation patient. PMID:26721826

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

    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)

  10. 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...

  11. Comparison of methods to differentiate staphylococcus and micrococcus species isolated from bovine mammary glands

    Directory of Open Access Journals (Sweden)

    Pengov A.

    2003-01-01

    Full Text Available The hemolytic pattern of colonies, the slide coagulase (clumping factor test, the tube coagulase test and the thermonuclease test were studied for their capability to differentiate Staphylococcus aureus from other Staphylococcus and Micrococcus species. A total 93 strains of Staphylococcus aureus isolated from bovine udder glands were tested. The hemolytic pattern of colonies and the slide coagulase test were found to be less reliable than other tests. No important differences could be found between the tube coagulase test and the thermonuclease test. The API-Staph test was used as a reference.

  12. A novel method of isolation of a bone-morphogenetic-protein-like protein from ossein.

    Science.gov (United States)

    Mythili, J; Padmavathy, S; Chandrakasan, G

    2001-08-01

    A new bone-morphogenetic-protein (BMP)-like protein has been isolated through a new protocol from a novel source, ossein. The BMP-like protein was hydrophilic and characterized through Fourier-transform IR studies, SDS/PAGE and coupled with a neutral binder, hydroxypropylmethylcellulose (HPMC) for control release. The IR spectrum of the protein showed peaks in tandem with BMP from bone matrix, and its molecular mass was in the range 18-21 kDa. Sustained release from the surface of HPMC was achieved for a period of 3 days. PMID:11483152

  13. Novel method for isolation of major phenolic constituents from cashew (Anacardium occidentale L.) nut shell liquid.

    Science.gov (United States)

    Paramashivappa, R; Kumar, P P; Vithayathil, P J; Rao, A S

    2001-05-01

    Commercially available cashew (Anacardium occidentale L.) nut shell liquid (CNSL) mainly contains the phenolic constituents anacardic acid, cardol, and cardanol. These phenolic constituents are themselves heterogeneous, and each of them contains saturated, monoene, diene, and trienes in the fifteen-carbon side chain. This communication describes the separation of anacardic acid, cardol, and cardanol for industrial application. Anacardic acid was selectively isolated as calcium anacardate. The acid-free CNSL was treated with liquor ammonia and extracted with hexane/ethyl acetate (98:2) to separate the mono phenolic component, cardanol. Subsequently, ammonia solution was extracted with ethyl acetate/hexane (80:20) to obtain cardol. PMID:11368634

  14. COMPARISON OF TWO METHODS OR DETECTION OF SECRETED ASPARTYL PROTEINASE IN URINARY ISOLATES OF CANDIDA SPECIES

    Directory of Open Access Journals (Sweden)

    Santosh Patil

    2014-04-01

    Results: Out of 150 candida species isolated from clinical urine samples 86.6% were C. tropicalis, followed by C. albicans (11.3%, C. glabrata (1.33% and C. dubliniensis (0.6%. SAP detection using Bovine serum agar was 29.3% where as using bovine haemoglobin agar was 18.6%. Conclusions: So we conclude that the bovine serum agar is far superior to bovine haemoglobin agar for detection of SAP. [Natl J Med Res 2014; 4(2.000: 119-121

  15. 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.)

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

    International Nuclear Information System (INIS)

    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 131I (131I-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 131I in yields of 95.6 ± 4.4% (n = 8), and in vivo studies showed that 131I-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.)

  17. 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.

  18. A robust and cost-effective method for DNA isolation from Satureja species (Lamiaceae

    Directory of Open Access Journals (Sweden)

    Dodoš Tanja

    2014-01-01

    Full Text Available Aromatic species of the genus Satureja are rich in secondary metabolites that interfere with DNA isolation procedures. Four protocols based on the standard CTDNA extraction protocol of Doyle and Doyle (1987 were tested in six savory taxa. The polyphenol adsorbents activated charcoal and/or polyvinylpyrrolidone 10 were employed in three procedures (B, C and D; for the elimination of polysaccharides, 4M NaCl was applied in the latter two. The highest DNA yield was obtained with Protocol D and averaged 1420.7±398.3 μg DNA/g of dry leaf tissue. Optimal values of the absorbance ratio 260/280 of all DNA solutions revealed the absence or only negligible contamination by proteins. Contamination by polysaccharides inferred from the absorbance ratio 260/230 showed that Protocol C provided the least contaminated material (average of 1.7±0.4. Enzymatic reactions of DNA solutions obtained by Protocol D showed amplification of both loci in all individuals. In conclusion, Protocol D is suitable for the isolation of high quantities of pure DNA from Satureja spp. [Projekat Ministarstva nauke Republike Srbije, br. 173029 i br. 173005

  19. Comparison of Rapid Centrifugation Assay with Conventional Tissue Culture Method for Isolation of Dengue 2 Virus in C6/36-HT Cells

    OpenAIRE

    Roche, Rosmari Rodríguez; Alvarez, Mayling; María G. Guzmán; Morier, Luis; Kourí, Gustavo

    2000-01-01

    A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results sug...

  20. Chemometric method of spectra analysis leading to isolation of lysozyme and CtDNA spectra affected by osmolytes.

    Science.gov (United States)

    Bruździak, Piotr; Rakowska, Paulina W; Stangret, Janusz

    2012-11-01

    In this paper we present a chemometric method of analysis leading to isolation of Fourier transform infrared (FT-IR) spectra of biomacromolecules (HEW lysozyme, ctDNA) affected by osmolytes (trimethylamine-N-oxide and N,N,N-trimethylglycine, respectively) in aqueous solutions. The method is based on the difference spectra method primarily used to characterize the structure of solvent affected by solute. The cyclical usage of factor analysis allows precise information to be obtained on the shape of "affected spectra" of analyzed biomacromolecules. "Affected spectra" of selected biomacromolecules give valuable information on their structure in the presence of the osmolytes in solution, as well as on the level of perturbation in dependence of osmolyte concentration. The method also gives a possibility of insight into the mechanism of interaction in presented types of systems. It can be easily adapted to various chemical and biochemical problems where vibrational or ultraviolet-visible (UV-Vis) spectroscopy is used. PMID:23146186

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs

    Directory of Open Access Journals (Sweden)

    Hall Leo T

    2004-03-01

    Full Text Available Abstract Background The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections. We aimed to isolate cDNAs of messages induced by switching Aspergillus nidulans from growth on glucose to growth on selected polysaccharides. Approximately 4,700 contigs from 12,320 ESTs were already available from a cDNA library representing transcripts isolated from glucose-grown A. nidulans during asexual development. Our goals were to expand the cDNA collection without repeated sequencing of previously identified ESTs and to find as many transcripts as possible that are specifically induced in complex polysaccharide metabolism. Results We have devised a Negative Subtraction Hybridization (NSH method and tested it in A. nidulans. NSH entails screening a plasmid library made from cDNAs prepared from cells grown under a selected physiological condition with labeled cDNA probes prepared from another physiological condition. Plasmids with inserts that failed to hybridize to cDNA probes through two rounds of screening (i.e. negatives indicate that they are transcripts present at low concentration in the labeled probe pool. Thus, these transcripts will be predominantly condition-specific, along with some rare transcripts. In a screen for transcripts induced by switching the carbon source from glucose to 12 selected polysaccharides, 3,532 negatives were isolated from approximately 100,000 surveyed colonies using this method. Negative clones were end-sequenced and assembled into 2,039 contigs, of which 1,722 were not present in the previously characterized glucose-grown cDNA library. Single-channel microarray hybridization experiments confirmed that the majority of the negatives represented genes that were differentially induced by a switch from growth in glucose to one or more of the polysaccharides

  6. 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

  7. Novel methods for accurate identification, isolation, and genomic analysis of symptomatic microenvironments in atherosclerotic arteries.

    Science.gov (United States)

    Slevin, Mark; Baldellou, Maribel; Hill, Elspeth; Alexander, Yvonne; McDowell, Garry; Murgatroyd, Christopher; Carroll, Michael; Degens, Hans; Krupinski, Jerzy; Rovira, Norma; Chowdhury, Mohammad; Serracino-Inglott, Ferdinand; Badimon, Lina

    2014-01-01

    A challenge facing surgeons is identification and selection of patients for carotid endarterectomy or coronary artery bypass/surgical intervention. While some patients with atherosclerosis develop unstable plaques liable to undergo thrombosis, others form more stable plaques and are asymptomatic. Identification of the cellular signaling mechanisms associated with production of the inflammatory, hemorrhagic lesions of mature heterogenic plaques will help significantly in our understanding of the differences in microenvironment associated with development of regions susceptible to rupture and thrombosis and may help to predict the risk of plaque rupture and guide surgical intervention to patients who will most benefit. Here, we demonstrate detailed and novel methodologies for successful and, more importantly, accurate and reproducible extraction, sampling, and analysis of micro-regions in stable and unstable coronary/carotid arteries. This information can be applied to samples from other origins and so should be useful for scientists working with micro-isolation techniques in all fields of biomedical science. PMID:24510873

  8. Investigation of autoionization spectra of Sm atoms using an isolated-core excitation method

    Institute of Scientific and Technical Information of China (English)

    Qin Wen-Jie; Dai Chang-Jian; Xiao Ying; Zhao Hong-Ying

    2009-01-01

    Using the isolated-core-excitation scheme and three-step laser resonance ionization spectroscopy approach, this paper, for the first time, has systematically investigated the autoionization spectra of atomic Sm, belonging to the 4f66pn/ and 4f55d6snl (l=0, 2) configurations. In the experiment, the first two tunable dye lasers are employed to excite the Srn atom from its initial state to the differcnt 4f66snl bound Rydberg states, then the third dye laser is scanned to drive the atom to the doubly-excited autoionizing states. With the above excitation scheme, the measured transition profiles of the autoionizing states are nearly symmetric, from which the level energies and widths can be easily obtained.

  9. Investigation of autoionization spectra of Sm atoms using an isolated-core excitation method

    Science.gov (United States)

    Qin, Wen-Jie; Dai, Chang-Jian; Xiao, Ying; Zhao, Hong-Ying

    2009-05-01

    Using the isolated-core-excitation scheme and three-step laser resonance ionization spectroscopy approach, this paper, for the first time, has systematically investigated the autoionization spectra of atomic Sm, belonging to the 4f66pnl and 4f55d6snl (l = 0,2) configurations. In the experiment, the first two tunable dye lasers are employed to excite the Sm atom from its initial state to the different 4f66snl bound Rydberg states, then the third dye laser is scanned to drive the atom to the doubly-excited autoionizing states. With the above excitation scheme, the measured transition profiles of the autoionizing states are nearly symmetric, from which the level energies and widths can be easily obtained.

  10. Fault detection, isolation and reconfiguration in FTMP Methods and experimental results. [fault tolerant multiprocessor

    Science.gov (United States)

    Lala, J. H.

    1983-01-01

    The Fault-Tolerant Multiprocessor (FTMP) is a highly reliable computer designed to meet a goal of 10 to the -10th failures per hour and built with the objective of flying an active-control transport aircraft. Fault detection, identification, and recovery software is described, and experimental results obtained by injecting faults in the pin level in the FTMP are presented. Over 21,000 faults were injected in the CPU, memory, bus interface circuits, and error detection, masking, and error reporting circuits of one LRU of the multiprocessor. Detection, isolation, and reconfiguration times were recorded for each fault, and the results were found to agree well with earlier assumptions made in reliability modeling.

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

    International Nuclear Information System (INIS)

    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

  12. Partial characterization of low density lipoprotein preparations isolated from fresh and frozen plasma after radiolabeling by seven different methods

    International Nuclear Information System (INIS)

    Four 99mTc and three 123I labeling methods were evaluated for their suitability to label low density lipoproteins (LDL) for the purpose of scintigraphic biodistribution studies. For 99mTc these methods were: direct incorporation in LDL of 99mTcO4- using sodium dithionite (dithionite method); a method using first N,N-dimethylformamide to prepare a 99mTc-complex reacting with LDL in a subsequent step (DMF method); a technique in which 99mTcO4- is first coupled to a diamide dithiolate derivative of pentanoic acid by reduction with dithionite, followed by coupling of this ligand to LDL (N2S2 method); and a method using sodium borohydride and stannous chloride as reducing agents (borohydride method). The iodination techniques were based on oxidation of I(-)----I+, using iodine monochloride (ICl method), 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (Iodogen method), and N-bromosuccinimide (NBS method) as oxidants. We studied labeling yields, modification of LDL caused by the labeling procedures using agarose-gel electrophoresis, and radiochemical stability of the labeled LDL complex upon incubation in plasma at 37 degrees C for 15 h. We used Sepharose CL6B chromatography to separate LDL from other plasma proteins. We also examined whether LDL isolated from frozen plasma (Pool-LDL) gave results similar to LDL obtained from freshly prepared plasma (Fresh-LDL). Pool-LDL radiolabeled by the dithionite, DMF, NBS, and Iodogen methods lost its label upon incubation with plasma. This also happened with Fresh-LDL when the DMF, NBS and Iodogen methods were used. Upon agarose-gel electrophoresis, no modification of LDL was observed with all methods when the radionuclide/LDL ratio was kept low

  13. Comparison of three methods for isolation of nucleic acids from membranate inner ear tissue of rats

    Institute of Scientific and Technical Information of China (English)

    KONG Wei-jia; WANG Ying; WANG Qiong; HAN Yue-chen; HU Yu-juan

    2006-01-01

    Background Mitochondrial DNA mutations have been found in sensorineural deafness. The aim of this study was to compare three methods for extraction of nucleic acid from membranate inner ear tissue of rats.Methods Alkaline denaturation, a conventional phenol-chloroform method and Trizol reagent were respectively used to extract the slight nucleic acid from membranate inner ear tissue of rats. We assessed the amount and quality of nucleic acid using a UV-spectrometer and polymerase chain reaction (PCR).Results The yield and purity (OD260/OD280) of DNA from inner ear tissue using the phenol-chloroform method was the highest of the three methods. Mitochondrial DNA (mtDNA) fragment can be amplified by PCR from nucleic acid prepared by all methods, while no nuclear DNA (nDNA) fragment can be amplified by method of alkaline denaturation. Both nuclear and mitochondrial genescould be amplified by reverse transcriptional PCR from the RNA prepared by Trizol reagent.Conclusion Adequate amount and high-quality of mtDNA, nDNA and RNA were obtained from unilateral membranate inner ear tissue of rats. Method of alkaline denaturation could be chosen when mtDNA without nDNA was needed, while phenol-chloroform method was suitable for extracting total DNA (including nDNA and mtDNA); method with Trizol reagent was suitable for extracting total RNA and total DNA.

  14. 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.

  15. Overexpressions of Lambda Phage Lysis Genes and Biosynthetic Genes of Poly-β-hydroxybutyrate in Recombinant E.coli

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    A plasmid (pTU9) containing the lambda (λ) phage lysis genes S(-)RRz and the biosynthetic genes phbCAB of poly-β-hydroxybutyrate (PHB) was constructed and transformed into E.coli JM109. Cultured in Luria-Bertani (LB) medium with 20 g/L glucose, E.coli JM109 (pTU9) could accumulate PHB in cells up to 40% (g PHB per g dry cells). A chelating agent EDTA was applied to induce a complete cell lysis and PHB granules were released. This method has a potential application in PHB separation.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-03-01

    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.

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

    International Nuclear Information System (INIS)

    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

  18. Condensed summary of the systems prioritization method as a decision-aiding approach for the waste isolation pilot plant

    International Nuclear Information System (INIS)

    In March 1994, the U.S. Department of Energy Carlsbad Area Office (DOE/CAO) implemented a performance-based decision-aiding method to assist in programme prioritization within the Waste Isolation Pilot Plant (WIPP) project. The prioritization was with respect to 40 CFR Part CFR Part 191, 13(a) and 450 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 combination of activities in terms of their predicted contributions 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. (Author)

  19. Application of Different Drying Methods on β-Glucan Isolated from Spent Brewer’s Yeast Using Alkaline Procedure

    Directory of Open Access Journals (Sweden)

    Vesna Zechner-krpan

    2010-03-01

    Full Text Available Water-insoluble (particulate β-glucan was isolated from the cell walls of spent brewer’s yeast using a single-step alkaline treatment. To stabilize β-glucan water suspensions, sonication was successfully applied. Three different drying methods were used: air-drying, lyophilization and spray-drying. Air-drying and lyophilization caused β-glucan particles agglomeration and changes of their microstructure. Sonication combined with spray-drying resulted in minimal β-glucan structural changes and negligible formation of agglomerates. Reaggregation of spray-dried β-glucan particles was minimal even after resuspending in water.

  20. Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii.

    Science.gov (United States)

    Ogasawara, Yasushi; Katayama, Kinya; Minami, Atsushi; Otsuka, Miyuki; Eguchi, Tadashi; Kakinuma, Katsumi

    2004-01-01

    Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well. PMID:15112997

  1. A red pigment synthesized by an Aspergillus parasiticus mutant as a possible new intermediate in the aflatoxin biosynthetic pathway.

    Science.gov (United States)

    García, M E; Herce, M D; Blanco, J L; Suárez, G

    1994-11-01

    The isolation of a red pigment from an Aspergillus parasiticus mutant obtained by 366 nm u.v. light treatment of A. parasiticus NRRL 2999 is described. Studies of conversion in aflatoxin B1 and G1 suggest that the red pigment could be a possible new intermediate in the aflatoxin biosynthetic pathway not described to date, and this has been verified by studies in gas chromatography/mass spectrometry. The solubility and stability characteristics under refrigeration storage, and the influence of the temperature and the pH on its production by the A. parasiticus mutant were also studied. It grew best at 30 degrees C and pH 6. The red pigment was most soluble in ethyl acetate. The results obtained in water are emphasized where there was high stability. PMID:8002480

  2. Effectiveness of a Simplified Method for Isolation of Burkholderia pseudomallei from Soil

    OpenAIRE

    Limmathurotsakul, Direk; Wuthiekanun, Vanaporn; Amornchai, Premjit; Wongsuwan, Gumphol; Day, Nicholas P. J.; Peacock, Sharon J.

    2012-01-01

    Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.

  3. Soft-landing ion deposition of isolated radioactive probe atoms on surfaces : A novel method

    NARCIS (Netherlands)

    Laurens, CR; Rosu, MF; Pleiter, F; Niesen, L

    1997-01-01

    We present a method to deposit a wide range of radioactive probe atoms on surfaces, without introducing lattice damage or contaminating the surface with other elements or isotopes. In this method, the probe atoms are mass separated using an isotope separator, decelerated to 5 eV, and directly deposi

  4. 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

  5. Optimisation of isolation methods for the azaspiracid group of marine biotoxins and the development of accurate and precise methods of analysis

    OpenAIRE

    Kilcoyle, J.

    2015-01-01

    The two main groups of biotoxins which affect the Irish shellfish industry are azaspiracids (AZAs) and the okadaic acid (OA) group (OA, DTX2, DTX1 and their esters) toxins. Since AZAs were first identified in 1998, well over 30 analogues have been reported. Structural and toxicological data have been described for AZA1–5 (isolated from shellfish). LC-MS/MS is the EU reference method for detection of the AZAs (AZA1, -2 and -3) and the OA group toxins in raw shellfish with the regulatory limit ...

  6. Optimisation of Isolation Methods for the AZA Group of Marine Biotoxins and the Development of Accurate and Precise Methods of Analysis

    OpenAIRE

    Kilcoyne, Jane

    2015-01-01

    The two main groups of biotoxins which affect the Irish shellfish industry are azaspiracids (AZAs) and the okadaic acid (OA) group (OA, DTX2, DTX1 and their esters) toxins. Since AZAs were first identified in 1998, well over 30 analogues have been reported. Structural and toxicological data have been described for AZA1–5 (isolated from shellfish). LC-MS/MS is the EU reference method for detection of the AZAs (AZA1, -2 and -3) and the OA group toxins in raw shellfish with the regulatory limit ...

  7. Effect of different isolation methods on structure and properties of lignin from valonea of Quercus variabilis.

    Science.gov (United States)

    Yang, Lina; Wang, Dongmei; Zhou, Dan; Zhang, Yawei

    2016-04-01

    Valonea of Quercus variabilis Blume, an abundant feedstock in China, can be used for tannin. However, there are little studies about lignin from this material. The present study aimed at lignin from the valonea: (1) Ethanol lignin (EL), alkali lignin (AL), milled wood lignin (MWL) and enzyme hydrolysis lignin (EHL) were prepared from the valonea of Q.variabilis Blume. (2) The effect of different isolation processes on the lignin chemical and physical features were studied by UV-vis, FT-IR, GPC, TG and (1)H NMR. (3) Antioxidant activities of four lignin preparations were evaluated by DPPH, ABTS and FRAP assays. The results showed that the valonea of Q. variabilis contained mass lignin and four lignin preparations were GSH-type with little differences. The MWL contained the least functional groups (1.75 mmol/g MeO, 0.87 mmol/g ArOH and 1.27 mmol/g AlkOH), the poorest thermostability (onset degradation temperature=111°C, maximum rate of degradation=268°C) and the highest antioxidant activity. The EHL had the highest molecular weight (Mw=1,429 g/mol; Mn=746.18 g/mol). This study provided a theoretical basis for the development and utilization of lignin from the valonea of Q. variabilis. PMID:26772919

  8. Determinação da origem biossintética de ácido acético através da técnica "Site Specific Natural Isotopic Fractionation Studied by Nuclear Magnetic Resonance (SNIF-NMR" Biosynthetic origin of acetic acid using SNIF-NMR

    Directory of Open Access Journals (Sweden)

    Elisangela Fabiana Boffo

    2006-06-01

    Full Text Available The main purpose of this work is to describe the use of the technique Site-Specific Natural Isotopic Fractionation of hydrogen (SNIF-NMR, using ²H and ¹H 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 C3, C4, and CAM biosynthetic mechanisms, blends of C3 and C4 (agrins and synthetic acetic acid.

  9. 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.

  10. Control channels with full galvanic isolation by the fiber optical communication line method

    International Nuclear Information System (INIS)

    Application of control channel with full galvanic insulation using the fiber optics communication line (FOCL) method was analyzed. The developed control systems provide full electrical insulation between the controlled facility and control center. Application of FOCL method excludes loops, occurring under conditions of coearthing of several pulse units, and background effects, occurring between control channels. The full insulation between the facility and control center assures the safety operation and high electric potential

  11. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

    Science.gov (United States)

    Rao, Pavitra N; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C; Carlton, Jane M

    2016-06-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  12. A simple HPLC method for the isolation and quantification of the allergens tuliposide A and tulipalin A in Alstroemeria.

    Science.gov (United States)

    Christensen, L P; Kristiansen, K

    1995-04-01

    A practical, rapid, reliable and sensitive method for the isolation and determination of the allergens tuliposide A and alpha-methylene-gamma-butyrolactone (tulipalin A), by reversed-phase high-performance liquid chromatography (RP-HPLC), has been developed in order to select Alstroemeria species for breeding purposes. From the aqueous extracts of flowers, stems and leaves, of several Alstroemeria species, the contents of 6-tuliposide A and tulipalin A were determined by isocratic RP-HPLC, using distilled water as mobile phase. The compounds were detected by an UV detector at 208 nm. Differences in 6-tuliposide A and tulipalin A content were found among the species investigated, with the highest concentrations in stems and flowers. The absence of other tuliposides (e.g., 1-tuliposide A, 1- and 6-tuliposide B) in extracts was proven by TLC, RP-HPLC, 1H- and 13C-NMR. 6-Tuliposide A and tulipalin A were identified by 1H- and 13C-NMR and comparison with authentic material, respectively. With this HPLC method, it is possible to investigate a large number of plants for their contents of tuliposide A and tulipalin A, within a minimum of time, and to isolate them directly from aqueous extracts. PMID:7600774

  13. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

    Science.gov (United States)

    Rao, Pavitra N.; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K.; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P.; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C.

    2016-01-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  14. Arginine and pyrimidine biosynthetic defects in Neisseria gonorrhoeae strains isolated from patients.

    OpenAIRE

    Shinners, E N; Catlin, B W

    1982-01-01

    Neisseria gonorrhoeae strains with nutritional requirements that include arginine (Arg-), uracil (Ura-), and hypoxanthine have attracted attention because of their tendency to cause disseminated infections, as a basis for genetic studies of arginine and pyrimidine biosynthesis, we examined the activities of four enzymes of these pathways in cell-free extracts of both prototrophic and Arg- Ura- strains. Activities of glutamate acetyltransferase, aspartate transcarbamylase, and orotate phosphor...

  15. Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds

    OpenAIRE

    Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa; Hirai, Itaru

    2015-01-01

    Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains ...

  16. Discordance across Phenotypic and Molecular Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Low TB Incidence Country

    Science.gov (United States)

    Ahmad, Suhail; Mokaddas, Eiman; Al-Mutairi, Noura; Eldeen, Hanaa S.; Mohammadi, Shirin

    2016-01-01

    With increasing incidence of multidrug-resistant tuberculosis (MDR-TB), accurate drug susceptibility testing (DST) of Mycobacterium tuberculosis to first-line drugs has become crucial for proper patient management. We evaluated concordance of DST results for 70 M. tuberculosis isolates across two phenotypic and two molecular methods: BACTEC 460TB, MGIT 960 system, GenoType MTBDRplus and DNA sequencing of gene segments most commonly implicated in conferring resistance to anti-TB drugs. Most (84%) M. tuberculosis isolates were multidrug-resistant. Twenty-four isolates yielded discrepant DST results. For rifampicin, isoniazid and streptomycin, 96%, 97% and 93% of isolates, respectively, were susceptible or resistant by all four methods, whereas for ethambutol, this agreement was observed for only 76% of isolates (P streptomycin versus ethambutol). Occurrence of rare mutations in three isolates that confer low-level resistance caused lower agreement for rifampicin among the four methods (kappa coefficient (κ) range, 0.84 to 0.95). For isoniazid, there was perfect agreement among phenotypic methods and molecular methods (κ, 1.00) but lower agreement between phenotypic and molecular methods. Three isolates were detected as polydrug-resistant by MGIT 960 system but as multidrug-resistant by DNA sequence-based method. The agreement was higher for streptomycin among the two phenotypic methods (κ, 0.97) while targeted sequencing yielded lower agreement (κ range, 0.86 to 0.89). The discrepancy for ethambutol resulted largely due to lower concordance of MGIT 960 results (κ range, 0.53 to 0.64). The MGIT 960 system is an accurate method for DST of M. tuberculosis against isoniazid and streptomycin while the results of rifampicin susceptibility should be complemented with DNA sequencing-based method when the suspicion for resistance is high. The possibility of false susceptibility to ethambutol with MGIT 960 system suggests that molecular or other phenotypic methods may be

  17. Ascaridia galli in chickens: intestinal localization and comparison of methods to isolate the larvae within the first week of infection.

    Science.gov (United States)

    Ferdushy, Tania; Nejsum, Peter; Roepstorff, Allan; Thamsborg, Stig M; Kyvsgaard, Niels C

    2012-12-01

    This study was conducted to observe the localization and to compare methods for isolation of minute Ascaridia galli larvae in chicken intestine. Firstly, six 7-week-old layer pullets were orally infected with 2,000 embryonated A. galli eggs and necropsied either at 3, 5 or 7 days post infection (dpi). More than 95 % of the recovered larvae were obtained from the anterior half of the jejunoileum, suggesting this part as the initial predilection site for A. galli larvae. Secondly, the intestinal wall of one layer pullet infected with 20,000 A. galli eggs 3 days earlier was digested in pepsin-HCl for 90 min. The initial 10 min of digestion released 51 % of the totally recovered larvae and the last 30 min of continuous digestion yielded only 5 %. This indicates that the majority of larvae were located superficially in the intestinal mucosa. Thirdly, 48 7-week-old layer pullets were infected with 500 A. galli eggs and necropsied at 3 dpi to compare three different larval isolation methods from the intestinal wall, viz., EDTA incubation, agar-gel incubation and pepsin-HCl digestion, resulting in mean percentages of the recovered larvae: 14.4, 18.2 and 20.0 %, respectively (P = 0.15). As conclusion, we recommended Pepsin-HCl digestion as the method of choice for larval recovery from the intestinal wall in future population dynamics study due to high efficiency and quick and simple detection. The agar-gel method was considered to be a prerequisite for molecular and immunological investigations as the larvae were more active and fully intact. PMID:22915270

  18. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    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.

  19. Characterization of sophorolipid biosynthetic enzymes from Starmerella bombicola.

    Science.gov (United States)

    Saerens, Karen M J; Van Bogaert, Inge N A; Soetaert, Wim

    2015-11-01

    Altering glycolipid structure by genetic engineering of Starmerella bombicola is a recently started research topic and worthy alternative to the unsuccessful selective feeding strategies conventionally applied to reach this goal. One question to be addressed when expressing heterologous proteins in S. bombicola is the activity of the subsequent biosynthetic enzymes toward such modified substrates. In this scope, we studied the substrate specificity of the UDP-glucosyltransferases UgtA1 and UgtB1, responsible for the stepwise synthesis of sophorolipids from a hydroxylated fatty acid, and that of the acetyltransferase, responsible for acetylation of the sophorolipid molecule. All enzymes showed specificity toward a C18:1 chained acceptor and both glucosyltransferases were highly selective toward the UDP-glucose donor. Severe product inhibition of the glucosyltransferases explains the limited accumulation of sophorolipid intermediates by earlier created single deletion mutants of S. bombicola. Finally, a more detailed study of the acetylation of sophorolipid intermediates sheds light on the enzymatic cascade during synthesis. PMID:26298016

  20. A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp.

    OpenAIRE

    Torres, R D; Oletta, C A; Zlotnik, H

    1996-01-01

    The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Ce...

  1. Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes

    Science.gov (United States)

    Cruz-Morales, Pablo; Kopp, Johannes Florian; Martínez-Guerrero, Christian; Yáñez-Guerra, Luis Alfonso; Selem-Mojica, Nelly; Ramos-Aboites, Hilda; Feldmann, Jörg; Barona-Gómez, Francisco

    2016-01-01

    Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored. Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded—repurposed enzyme families—from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy. As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real ‘chemical dark matter’ will be unveiled. PMID:27289100

  2. Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes.

    Science.gov (United States)

    Cruz-Morales, Pablo; Kopp, Johannes Florian; Martínez-Guerrero, Christian; Yáñez-Guerra, Luis Alfonso; Selem-Mojica, Nelly; Ramos-Aboites, Hilda; Feldmann, Jörg; Barona-Gómez, Francisco

    2016-01-01

    Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored.Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded-repurposed enzyme families-from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy.As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real 'chemical dark matter' will be unveiled. PMID:27289100

  3. Coordinated regulation of biosynthetic and regulatory genes coincides with anthocyanin accumulation in developing eggplant fruit

    Science.gov (United States)

    Violet to black pigmentation of eggplant (Solanum melongena) fruit is attributed to anthocyanin accumulation. Model systems support the interaction of biosynthetic and regulatory genes for anthocyanin biosynthesis. Anthocyanin structural gene transcription requires the expression of at least one m...

  4. Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria

    OpenAIRE

    Blodgett, Joshua A. V.; Oh, Dong-Chan; Cao, Shugeng; Currie, Cameron R.; Kolter, Roberto; Clardy, Jon

    2010-01-01

    A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identificati...

  5. 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

    that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each...... 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....

  6. A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol.

    Science.gov (United States)

    Likhite, Neah; Warawdekar, Ujjwala M

    2011-04-01

    The aim of this study was to develop a systems approach to study tumor tissue. The importance of concurrent extraction of RNA, DNA, and protein is evident when genetic aberrations and the differences in the proteome and transcriptome have to be correlated. The need is magnified, as the tissue available for study is miniscule, is shared amongst investigators, and needs to support the holistic approach. Trizol is a monophasic solution of phenol and guanidine isothiocyanate and can be used to isolate the three biomolecules simultaneously. Trizol solution was used for RNA extraction in an ongoing study about expression of molecular markers in non-small cell lung carcinoma (NSCLC) and breast tumor tissue. After isolation of RNA, the remaining Trizol fraction was stored at -80°C for over 6 months. We have shown the extraction of protein from 17 tumor and adjacent, normal tissue samples and PBMC obtained from four blood samples. The isolation and solubilization of the protein fraction were done according to the product information using isopropanol for precipitation and guanidine hydrochloride and SDS for washing and solubilization, respectively, modifying the time of solubilization. The protein was estimated by the bicinchoninic acid (BCA) method and analyzed on polyacrylamide gels. Staining showed a wide repertoire, and Western blotting confirmed extraction of cytokeratins (CK) and DNA repair proteins. Whereas tissue samples in which the RNA was degraded could be assessed by the presence of the protein salvaging the marker analysis, it was seen that nuclear proteins cannot be retrieved and are probably lost with the DNA fraction. PMID:21455480

  7. Effects of processing methods on composition and functionality of volatile components isolated from immature fruits of atemoya.

    Science.gov (United States)

    Liu, Tai-Ti; Chao, Louis Kuo-Ping; Peng, Chi-Wei; Yang, Tsung-Shi

    2016-07-01

    Atemoya is one of the most important commercial fruits of the family Annonaceae. The immature fruits of atemoya amply produced from a fruit-thinning process is normally regarded as waste and discarded. This research aimed at studying antimicrobial, antioxidant, and anti-inflammatory activities of the essential oil (EO) isolated from the immature fruits to explore its potential application. The fruits were subjected to different drying methods: solar drying (SD), oven drying at 30°C (OD-30), and at 50°C (OD-50). The oven drying method gave a higher EO yield than the solar drying method. Spathulenol was the largest compound in the EO after the drying process. Antimicrobial effect was not affected by the different drying methods. Antioxidant activity of the EO was measured by DPPH, nitric oxide, and reducing power methods. The EOOD-50 exhibited a stronger antioxidant activity than EOSD and EOOD-30. The EO also showed an anti-inflammatory activity in a cell model. PMID:26920282

  8. An improved radiochemical method for the isolation of uranium from other actinides in urine

    International Nuclear Information System (INIS)

    The Bioassay Laboratory at CRNL estimates uranium in urine by extraction with TBP (tributyl phosphate) and alpha counting. Studies performed on this method revealed several disadvantages: other actinides follow uranium and the overall recovery of uranium is not high. Cross-contamination studies showed that plutonium-239, neptunium-237, thorium-natural and curium-244 were all present in the final uranium source (Pu-239, 60 percent; Np-237, 48 percent; Th-nat, 40 percent; Cm-244, 15 percent). The overall uranium recovery was about 60 percent. The method has since been modified to improve the uranium recovery to 85 +- 5 percent and completely eliminate actinide cross-contamination by the inclusion of some selective co-precipitation steps after the TBP extractions have been carried out

  9. 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.

  10. 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 not...... 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....

  11. Improved method of isolating bacteria from joint fluids by the use of blood culture bottles.

    OpenAIRE

    von Essen, R; Hölttä, A

    1986-01-01

    An analysis of 47 episodes of bacterial arthritis showed that applying a blood culture procedure to the culture of joint fluids gave positive results in 10 cases (21%) that were negative by conventional methods. When follow up samples taken during antibiotic treatment were considered the proportion of false negatives eliminated rose to 40%. The respective advantages of using a large volume of inoculum and a large volume of medium are that very low numbers of viable bacteria in infected fluid ...

  12. Comparison of Extraction Methods for Isolation of 20-Hydroxyecdysone from Leuzea carthamoides

    Czech Academy of Sciences Publication Activity Database

    Machalová, Zdeňka; Sajfrtová, Marie; Sovová, Helena; Bártlová, Milena

    Prague : Orgit, 2014, s. 45. ISBN 978-80-02-02555-9. [International Congress of Chemical and Process Engineering /21./ - CHISA 2014 and Conference on Process Integration, Modelling and Optimisation for Energy Saving and Pollution Reduction /17./ - PRES 2014. Prague (CZ), 23.08.2014-27.08.2014] R&D Projects: GA TA ČR TA01010578 Institutional support: RVO:67985858 Keywords : extraction methods * pressurized liquid extraction * supercritical fluids extraction Subject RIV: CI - Industrial Chemistry, Chemical Engineering

  13. Comparison of Extraction Methods for Isolation of 20-Hydroxyecdysone from Leuzea carthamoides

    Czech Academy of Sciences Publication Activity Database

    Machalová, Zdeňka; Sajfrtová, Marie; Sovová, Helena; Bártlová, Milena

    Prague: Orgit, 2014, s. 45. ISBN 978-80-02-02555-9. [International Congress of Chemical and Process Engineering /21./ - CHISA 2014 and Conference on Process Integration, Modelling and Optimisation for Energy Saving and Pollution Reduction /17./ - PRES 2014. Prague (CZ), 23.08.2014-27.08.2014] R&D Projects: GA TA ČR TA01010578 Institutional support: RVO:67985858 Keywords : extraction methods * pressurized liquid extraction * supercritical fluids extraction Subject RIV: CI - Industrial Chemistry, Chemical Engineering

  14. Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust.

    OpenAIRE

    Treuhaft, M W; Arden Jones, M P

    1982-01-01

    Thermophilic actinomycetes are the primary sensitizing agents in farmer's lung disease. We compared dilution pour-plate and spread-plate methods for their usefulness in enumerating thermophilic actinomycetes in moldy silage dust and evaluated the ability of a nonquantitative gravity settling technique to recover thermophilic actinomycetes from moldy silage. Spread plates and pour plates yielded similar estimates of total thermophiles. Higher counts were observed on spread plates (P less than ...

  15. Adaptive methods for on-line recognition of isolated handwritten characters

    OpenAIRE

    Vuori, Vuokko

    2002-01-01

    The main goal of the work presented in this thesis has been the development of an on-line handwriting recognition system which is able to recognize handwritten characters of several different writing styles and is able to improve its performance by adapting itself to new writing styles. The recognition method should be applicable to hand-held devices of limited memory and computational resources. The adaptation process should take place during normal use of the device, not in some specific tr...

  16. Method of targeted delivery of laser beam to isolated retinal rods by fiber optics

    OpenAIRE

    Sim, Nigel; Bessarab, Dmitri; Jones, C. Michael; Krivitsky, Leonid

    2011-01-01

    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.

  17. Assessment of molecular methods as a tool for detecting pathogenic protozoa isolated from water bodies.

    Science.gov (United States)

    Adamska, M; Sawczuk, M; Kolodziejczyk, L; Skotarczak, B

    2015-12-01

    Several species belong to the Cryptosporidium and Giardia genus, the main parasitic protozoa occurring in water, but only some of them are infectious to humans. We investigated the occurrence of Cryptosporidium and Giardia and identified their species in the water samples collected from natural water bodies in north-western Poland. A total of 600 samples from water bodies used for bathing, sewage discharge, as drinking water sources and watering places for animals were screened. The samples were collected during a 3-year period in each of the four seasons and filtered using Filta-Max (IDEXX Laboratories, USA). Genomic DNA was extracted from all samples and used as a target sequence for polymerase chain reaction (PCR) and TaqMan real-time PCR, as well as for reverse line blotting (RLB) methods. PCR methods seem to be more sensitive to detect Giardia and Cryptosporidium DNA in water samples than RLB methods. All PCR products were sequenced and three were identified as C. parvum and four as G. intestinalis. The overall prevalence of C. parvum (0.5%) and G. intestinalis (0.6%) in the samples suggests that the risk of Cryptosporidium and Giardia infections in north-western Poland is minimal. PMID:26608757

  18. Differential effect of environmental conditions on the growth and regulation of the fumonisin biosynthetic gene FUM1 in the maize pathogens and fumonisin producers Fusarium verticillioides and Fusarium proliferatum

    OpenAIRE

    Marin, P.; Magan, Naresh; Vazquez, C.; Gonzalez-Jaen, M. T.

    2010-01-01

    The effects of ecophysiological factors, temperature and solute potential, on both the growth and the regulation of the fumonisin biosynthetic FUM1 gene were studied and compared in one isolate each of the two closely related fumonisin- producing and maize pathogens Fusarium verticillioides and Fusarium proliferatum. The effect of solute potential and temperature was examined on in vitro mycelia growth and on the expression of the FUM1 gene, quantified by species-specific re...

  19. 1. Improving the Yield of Biodiesel from Microalgae and Other Lipids. 2. Studies of the Wax Ester Biosynthetic Pathway and Potential Biotechnological Application

    OpenAIRE

    Wahlen, Bradley D.

    2012-01-01

    The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels. Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain ...

  20. Use of metabolic and molecular methods for the identification of a Bacillus strain isolated from paper affected by foxing.

    Science.gov (United States)

    De Paolis, Maria Rita; Lippi, Daniela

    2008-01-01

    Foxing of paper is a deterioration phenomenon occurring in the form of brown-yellowish spots, the abiotic and/or biotic causes of which are not yet completely understood. Nevertheless, microbiological infection has been recognized that may contribute to paper damage and therefore it becomes important to know the taxonomic position and the degradative activity of the potential infectious biological agents which mostly are fungi, but also bacteria and yeasts. A cellulolytic bacterial strain isolated from a foxed paper sample exhibited morphological and physiological characteristics of the Bacillus genus. To study its taxonomic position, different identification methods were used: the Biolog system, the direct amplified polymorphic DNA-polymerase chain reaction analysis (DAPD-PCR) and the partial sequencing of the 16S rDNA gene. Biolog system and partial sequencing of 16S rDNA gene assigned the strain to the Paenibacillus polymyxa species. DAPD-PCR analysis indicated a high similarity with Bacillus circulans, by comparing the isolated strain with some closely related Bacillus species. PMID:17686620

  1. Analysis of Genetic Similarities Between Clostridium Perfringens Isolates Isolated from Patients with Gas Gangrene and from Hospital Environment Conducted with the use of the PFGE Method

    Directory of Open Access Journals (Sweden)

    Brzychczy-Włoch Monika

    2014-03-01

    Full Text Available The objective of the study was to perform a comparative analysis of genetic similarity, with the use of pulsed field gel electrophoresis (PFGE, of Clostridium perfringens isolates originating from patients with gas gangrene and from the hospital environment. The study encompassed two patients with a clinical and microbiological diagnosis of gas gangrene, who were hospitalized in one of the hospitals of the Małopolska province in the time period between 31st March 2012 and 18th May 2012. Clostridium perfringens isolates genotyping indicated that the isolates originating from the two studied patients did not display genetic similarity and represented two different PFGE types, which corresponded to two different clones (clone A and B. Whereas the strains isolated from the hospital environment were genetically identical with the strain coming from the second patient and represented one PFGE type, which corresponded to one clone (clone A. As a result of the study, it is possible to conclude that both patients developed endogenous infection. Even so, the examination of the hospital environment indicates the possibility of the appearance of exogenous infections. It prompts recommending and following the exact regulations of sanitary regime in the ward and the operating theater if a patient is diagnosed with gas gangrene.

  2. 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.

  3. An expedited method for isolation of DNA for PCR from Magnaporthe oryzae stored on filter paper

    Institute of Scientific and Technical Information of China (English)

    Yulin; Jia; Yeshi; A.; Wamishe; Bo; Zhou

    2014-01-01

    The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 °C.Inoculated filter papers are cut into pieces of 0.5–1.0 cm diameter prior to storage. In the present study, a fast(11 min) and simple method of preparing DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction(PCR) was developed. A piece of filter paper containing the fungus was removed from a glass bottle and placed in a 0.2 mL Eppendorf tube containing 100 μL 10 × TE. The suspension was heated for 10 min at 95 °C in a PCR machine. The tube was then centrifuged for 1 min at 3000 r min-1. One μL of 10 × TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using a conventional DNA preparation method. DNA samples obtained from both methods were stored at 4 °C. PCR was performed with DNA on the preparation day and after 4, 8,10, and 18 days of refrigerated storage. In four samples, samples 12, 13, 14, and 28, AVR-Pi9 failed to be amplified. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1, confirming that the quality of the samples was insufficient for PCR. Overall, for nearly 90%(24/28) of the samples, the quality of the DNA prepared directly from the fungus on filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR.This method will be useful and effective for reducing cost and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.

  4. An expedited method for isolation of DNA for PCR from Magnaporthe oryzae stored on filter paper

    Directory of Open Access Journals (Sweden)

    Yulin Jia

    2014-10-01

    Full Text Available The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 °C. Inoculated filter papers are cut into pieces of 0.5–1.0 cm diameter prior to storage. In the present study, a fast (11 min and simple method of preparing DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction (PCR was developed. A piece of filter paper containing the fungus was removed from a glass bottle and placed in a 0.2 mL Eppendorf tube containing 100 μL 10 × TE. The suspension was heated for 10 min at 95 °C in a PCR machine. The tube was then centrifuged for 1 min at 3000 r min− 1. One μL of 10 × TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using a conventional DNA preparation method. DNA samples obtained from both methods were stored at 4 °C. PCR was performed with DNA on the preparation day and after 4, 8, 10, and 18 days of refrigerated storage. In four samples, samples 12, 13, 14, and 28, AVR-Pi9 failed to be amplified. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1, confirming that the quality of the samples was insufficient for PCR. Overall, for nearly 90% (24/28 of the samples, the quality of the DNA prepared directly from the fungus on filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR. This method will be useful and effective for reducing cost and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.

  5. AN IMPROVED METHOD FOR ISOLATING RESIDUAL KRAFT LIGNIN IN HIGH YIELD AND PURITY

    Institute of Scientific and Technical Information of China (English)

    Shubin Wu; Dimitris S.Argyropoulos

    2004-01-01

    When washed pulps is milled and ground to a fine powder, the resulting material may easily be degraded by cellulolytic enzymes. The klason and UV lignin content of the solid residuals obtained in this step were 49.9% lignin for spruce KP, and 21.4% for poplar KP. The solid residuals from enzymatic treatment contained about 93.3% and 90.7% of the lignin originally presented in the spruce KP and poplar KP respectively. The enzymatic treated residual was then subjected to mild acidolysis, which caused the cleavage of lignin-carbohydrate linkages.The resulting Ground Enzymatic/Acidolysis Kraft Lignin (GEA-KL) is of significantly higher yield than our previous two-step (enzymatic/acidolysis) residual kraft lignin (EA-KL). The improved method offers kraft lignin preparations in higher yield and purity than any other known method with minimal work up and solvent requirements. DFRC/quantitative 31p NMR protocol and quantitative DEPT edited 13C RMR were used for characterizing of RKLs.

  6. AN IMPROVED METHOD FOR ISOLATING RESIDUAL KRAFT LIGNIN IN HIGH YIELD AND PURITY

    Institute of Scientific and Technical Information of China (English)

    ShubinWu; DimitrisS.Argyropoulos

    2004-01-01

    When washed pulps is milled and ground to a fine powder, the resulting material may easily be degraded by cellulolytic enzymes. The klason and UV lignin content of the solid residuals obtained in this step were 49.9% lignin for spruce KP, and 21.4% for poplar KP. The solid residuals from enzymatic treatment contained about 93.3% and 90.7% of the lignin originally presented in the spruce KP and poplar KP respectively. The enzymatic treated residual was then subjected to mild acidolysis, which caused the cleavage of lignin-carbohydrate linkages. The resulting Ground Enzymatic/Acidolysis KraftLignin (GEA-KL) is of significantly higher yield than our previous two-step (enzymatic/acidolysis) residual kraft lignin (EA-KL). The improved method offers kraft lignin preparations in higher yield and purity than any other known method with minimal work up and solvent requirements. DFRC/quantitative P NMR protocol and quantitative DEPT edited C RMR were used for characterizing of RKLs.

  7. 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.

  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]. E-mail: giba_04@yahoo.com.br

    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 an effective and efficient DNA isolation method for Cinnamomum species.

    Science.gov (United States)

    Bhau, B S; Gogoi, G; Baruah, D; Ahmed, R; Hazarika, G; Ghosh, S; Borah, B; Gogoi, B; Sarmah, D K; Nath, S C; Wann, S B

    2015-12-01

    Different species of Cinnamomum are rich in polysaccharide's and secondary metabolites, which hinder the process of DNA extraction. High quality DNA is the pre-requisite for any molecular biology study. In this paper we report a modified method for high quality and quantity of DNA extraction from both lyophilized and non-lyophilized leaf samples. Protocol reported differs from the CTAB procedure by addition of higher concentration of salt and activated charcoal to remove the polysaccharides and polyphenols. Wide utility of the modified protocol was proved by DNA extraction from different woody species and 4 Cinnamomum species. Therefore, this protocol has also been validated in different species of plants containing high levels of polyphenols and polysaccharides. The extracted DNA showed perfect amplification when subjected to RAPD, restriction digestion and amplification with DNA barcoding primers. The DNA extraction protocol is reproducible and can be applied for any plant molecular biology study. PMID:26041191

  10. Electrically isolated, high melting point, metal wire arrays and method of making same

    Energy Technology Data Exchange (ETDEWEB)

    Simpson, John T.; Cunningham, Joseph P.; D' Urso, Brian R.; Hendricks, Troy R.; Schaeffer, Daniel A.

    2016-01-26

    A method of making a wire array includes the step of providing a tube of a sealing material and having an interior surface, and positioning a wire in the tube, the wire having an exterior surface. The tube is heated to soften the tube, and the softened tube is drawn and collapsed by a mild vacuum to bring the interior surface of the tube into contact with the wire to create a coated wire. The coated wires are bundled. The bundled coated wires are heated under vacuum to fuse the tube material coating the wires and create a fused rod with a wire array embedded therein. The fused rod is cut to form a wire array. A wire array is also disclosed.

  11. 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 mainstream...... procedure that allows users to assess which model (e.g. with or without an environment effect) is most suited. We code all our MCMC algorithms in a mix of compiled languages which allows us to decrease computing time by at least one order of magnitude. We propose an approximate inference and model selection...... 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/....

  12. Geometric spin frustration for isolated plaquettes of the lattices: An extended irreducible tensor operator method

    International Nuclear Information System (INIS)

    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

  13. Determinação da origem biossintética de ácido acético através da técnica "Site Specific Natural Isotopic Fractionation Studied by Nuclear Magnetic Resonance (SNIF-NMR)" Biosynthetic origin of acetic acid using SNIF-NMR

    OpenAIRE

    Elisangela Fabiana Boffo; Antonio Gilberto Ferreira

    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 ²H and ¹H 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 me...

  14. 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

    Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules....... 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 of the...... different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols...

  15. A simple wax-embedding method for isolation of aphid hemolymph for detection of luteoviruses in the hemocoel.

    Science.gov (United States)

    Liu, Sijun; Bonning, Bryony C; Allen Miller, W

    2006-03-01

    A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid. Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus (BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively. A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and their insect vectors. PMID:16307802

  16. Influence of different susceptibility testing methods and media on determination of the relevant fluconazole minimum inhibitory concentrations for heavy trailing Candida isolates with low-high phenotype.

    Science.gov (United States)

    Alp, Sehnaz; Sancak, Banu; Hascelik, Gulsen; Arikan, Sevtap

    2010-11-01

    We investigated the incidence of trailing growth with fluconazole in 101 clinical Candida isolates (49 C. albicans and 52 C. tropicalis) and tried to establish the convenient susceptibility testing method and medium for fluconazole minimum inhibitory concentration (MIC) determination. MICs were determined by CLSI M27-A2 broth microdilution (BMD) and Etest methods on RPMI-1640 agar supplemented with 2% glucose (RPG) and on Mueller-Hinton agar supplemented with 2% glucose and 0.5 μg ml(-1) methylene blue (GMB). BMD and Etest MICs were read at 24 and 48 h, and susceptibility categories were compared. All isolates were determined as susceptible with BMD, Etest-RPG and Etest-GMB at 24 h. While all isolates were interpreted as susceptible at 48 h on Etest-RPG and Etest-GMB, one C. albicans isolate was interpreted as susceptible-dose dependent (S-DD) and two C. tropicalis isolates were interpreted as resistant with BMD. On Etest-RPG, trailing growth caused widespread microcolonies within the inhibition zone and resulted in confusion in MIC determination. On Etest-GMB, because of the nearly absence of microcolonies within the zone of inhibition, MICs were evaluated more easily. We conclude that, for the determination of fluconazole MICs of trailing Candida isolates, the Etest method has an advantage over BMD and can be used along with this reference method. Moreover, GMB appears more beneficial than RPG for the fluconazole Etest. PMID:19563491

  17. 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.

  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. Isolation and Damping of Shocks, Vibrations and Seismic Movements at Buildings: Equipment and Pipe Networks by SERB-SITON Method

    International Nuclear Information System (INIS)

    The paper presents SERB -- SITON method to control, limit and damp the shocks, vibration, impact load and seismic movements with applications in buildings, equipment and pipe networks (herein called: 'components'). The elimination or reduction of shocks, vibration, impact load and seismic movements is a difficult problem, still improperly handled theoretically and practically because many times the phenomena are random in character and the behavior of components is non-linear with variations of the properties in time, variations that lead to the increase or decrease of the energy and impulse transfer from the dynamic excitation to the components. Moreover, the existing supports and dampers applied today, are not efficient enough in the reduction of the dynamic movement for all the frequency ranges met with in the technical application field. The stiffness and damping of classic supports do not allow a good isolation of components against shocks and vibrations so to eliminate their propagation to the environment and neither do they provide a satisfactory protection of the components sensitive to shocks and vibrations and seismic movements coming from the environment. In order to reduce the effects of shocks, vibrations impact and seismic movements on the components, this paper presents the results obtained by SITON on the concept, design, construction, experimental testing and application of new types of supports, devices and thin lattice structure, called 'SERB', capable to overtake large static loads, to allow displacements from impact, thermal expansions or yielding of supports and which, in any work position, can elastically overtake large dynamic loads or impact loads which they damp. The new supports and devices and thin lattice structure allow their adjustment without the occurrence of over-stressing in the components due to their non -- linear geometric behavior, and the contact pressure among the elements is limited to pre-set values to avoid blocking

  20. ORIGINAL ARTICLE: Detection of β-Lactamase Activity in Various Clinical Bacterial Isolates by Three Different Methods and its Correlation with Drug Resistance.

    Directory of Open Access Journals (Sweden)

    Sanjay M Wavare

    2012-07-01

    Full Text Available Background: β-lactams such as penicillins are the most widely used antibiotics, and β-lactamases are the greatest source of resistance to penicillins. Aims and Objectives: To study β-lactamase production in clinical isolates of family Enterobacteriaceae, P. aeruginosa and Staphylococci by three different methods and to correlate its potential with drug resistance; with an endeavour to evaluate convenient and economical method duly supported by relevant Minimum Inhibitory Concentration (MIC studies. Material and Methods: Total 240 clinical isolates (Gram-negative bacilli-191, staphylococci-49 were subjected to antimicrobial susceptibility testing by Kirby-Bauer disk diffusion method and MIC for ampicillin and penicillin was determined by agar dilution method. β-lactamase was detected by broth acidometric, iodometric cell suspension and microbiological method. Results: Multidrug resistance was observed in more than 90% isolates. One hundred and ninety Gram-negative bacilli were resistant to ampicillin and 47 staphylococcal isolates were resistant to both penicillin and ampicillin. Though microbiological method gave highest positive results 210 (87.5%, iodometric method could detect β-lactamase in apparently sensitive isolates as well giving satisfactory [207 (86.25%] comparable results. Conclusion: In view of the noted bacterial resistance, tests for β-lactamase should be carried out on a routine basis for an early implementation of appropriate antimicrobial therapy. Iodometric method is eminently convenient, economical and reliable method. Isolates showing MIC <0.125µg/ml for penicillin and MIC <8µg/ml for ampicillin should be checked for β-lactamase production.

  1. The raman spectrum of biosynthetic human growth hormone. Its deconvolution, bandfitting, and interpretation

    Science.gov (United States)

    Tensmeyer, Lowell G.

    1988-05-01

    The Raman spectrum of amorphous biosynthetic human growth hormone, somatotropin, has been measured at high signal-to-noise ratios, using a CW argon ion laser and single channel detection. The rms signal-to-noise ratio varies from 1800:1 in the Amide I region near 1650 cm -1 region, to 500:1 in the disulfide stretch region near 500 cm -1. Component Raman bands have been extracted from the entire spectral envelope from 1800-400 cm -1, by an interactive process involving both partial deconvolution and band-fitting. Interconsistency of all bands has been achieved by multiple overlapping of adjacent regions that had been isolated for the band-fitting programs. The resulting areas of the Raman component bands have been interpreted to show the ratios of peptide conformations in the hormone: 64% α-helix, 24% β-sheet, 8% β-turns and 4% γ-turns. Analysis of the tyrosine region, usually described as a Fermi resonance doublet near ˜830-850 cm -1, shows four bands, at 825, 833, 853, and 859 cm -1 in this macromolecule. Integrated intensities of these bands (2:2:2:2) are interpreted to show that only half of the eight tyrosine residues function as hydrogen-bond bridges via the acceptance of protons. Both disulfide bridges fall within the frequency ranges for normal, unstressed SS bonds: The 511 and 529 cm -1 bands are indicative of the gauche-gauche-gauche and trans-gauche-gauche conformations, respectively.

  2. Nutritional regulation of long-chain PUFA biosynthetic genes in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Gregory, Melissa K; Collins, Robert O; Tocher, Douglas R; James, Michael J; Turchini, Giovanni M

    2016-05-01

    Most studies on dietary vegetable oil in rainbow trout (Oncorhynchus mykiss) have been conducted on a background of dietary EPA (20 : 5n-3) and DHA (22 : 6n-3) contained in the fishmeal used as a protein source in aquaculture feed. If dietary EPA and DHA repress their endogenous synthesis from α-linolenic acid (ALA, 18 : 3n-3), then the potential of ALA-containing vegetable oils to maintain tissue EPA and DHA has been underestimated. We examined the effect of individual dietary n-3 PUFA on the expression of the biosynthetic genes required for metabolism of ALA to DHA in rainbow trout. A total of 720 juvenile rainbow trout were allocated to twenty-four experimental tanks and assigned one of eight diets. The effect of dietary ALA, EPA or DHA, in isolation or in combination, on hepatic expression of fatty acyl desaturase (FADS)2a(Δ6), FADS2b(Δ5), elongation of very long-chain fatty acid (ELOVL)5 and ELOVL2 was examined after 3 weeks of dietary intervention. The effect of these diets on liver and muscle phospholipid PUFA composition was also examined. The expression levels of FADS2a(Δ6), ELOVL5 and ELOVL2 were highest when diets were high in ALA, with no added EPA or DHA. Under these conditions ALA was readily converted to tissue DHA. Dietary DHA had the largest and most consistent effect in down-regulating the gene expression of all four genes. The ELOVL5 expression was the least responsive of the four genes to dietary n-3 PUFA changes. These findings should be considered when optimising aquaculture feeds containing vegetable oils and/or fish oil or fishmeal to achieve maximum DHA synthesis. PMID:26987422

  3. Analysis of Genetic Similarities Between Clostridium Perfringens Isolates Isolated from Patients with Gas Gangrene and from Hospital Environment Conducted with the use of the PFGE Method

    OpenAIRE

    Brzychczy-Włoch Monika; Bulanda Małgorzata

    2014-01-01

    The objective of the study was to perform a comparative analysis of genetic similarity, with the use of pulsed field gel electrophoresis (PFGE), of Clostridium perfringens isolates originating from patients with gas gangrene and from the hospital environment. The study encompassed two patients with a clinical and microbiological diagnosis of gas gangrene, who were hospitalized in one of the hospitals of the Małopolska province in the time period between 31st March 2012 and 18th May 2012. Clos...

  4. Multicenter Evaluation of Clinical Diagnostic Methods for Detection and Isolation of Campylobacter spp. from Stool.

    Science.gov (United States)

    Fitzgerald, Collette; Patrick, Mary; Gonzalez, Anthony; Akin, Joshua; Polage, Christopher R; Wymore, Kate; Gillim-Ross, Laura; Xavier, Karen; Sadlowski, Jennifer; Monahan, Jan; Hurd, Sharon; Dahlberg, Suzanne; Jerris, Robert; Watson, Renee; Santovenia, Monica; Mitchell, David; Harrison, Cassandra; Tobin-D'Angelo, Melissa; DeMartino, Mary; Pentella, Michael; Razeq, Jafar; Leonard, Celere; Jung, Carrianne; Achong-Bowe, Ria; Evans, Yaaqobah; Jain, Damini; Juni, Billie; Leano, Fe; Robinson, Trisha; Smith, Kirk; Gittelman, Rachel M; Garrigan, Charles; Nachamkin, Irving

    2016-05-01

    The use of culture-independent diagnostic tests (CIDTs), such as stool antigen tests, as standalone tests for the detection of Campylobacter in stool is increasing. We conducted a prospective, multicenter study to evaluate the performance of stool antigen CIDTs compared to culture and PCR for Campylobacter detection. Between July and October 2010, we tested 2,767 stool specimens from patients with gastrointestinal illness with the following methods: four types of Campylobacter selective media, four commercial stool antigen assays, and a commercial PCR assay. Illnesses from which specimens were positive by one or more culture media or at least one CIDT and PCR were designated "cases." A total of 95 specimens (3.4%) met the case definition. The stool antigen CIDTs ranged from 79.6% to 87.6% in sensitivity, 95.9 to 99.5% in specificity, and 41.3 to 84.3% in positive predictive value. Culture alone detected 80/89 (89.9% sensitivity) Campylobacter jejuni/Campylobacter coli-positive cases. Of the 209 noncases that were positive by at least one CIDT, only one (0.48%) was positive by all four stool antigen tests, and 73% were positive by just one stool antigen test. The questionable relevance of unconfirmed positive stool antigen CIDT results was supported by the finding that noncases were less likely than cases to have gastrointestinal symptoms. Thus, while the tests were convenient to use, the sensitivity, specificity, and positive predictive value of Campylobacter stool antigen tests were highly variable. Given the relatively low incidence of Campylobacter disease and the generally poor diagnostic test characteristics, this study calls into question the use of commercially available stool antigen CIDTs as standalone tests for direct detection of Campylobacter in stool. PMID:26962088

  5. 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.

  6. The feasibility of isolation and detection of fullerenes and carbon nanotubes using the benzene polycarboxylic acid method

    International Nuclear Information System (INIS)

    The incorporation of fullerenes and carbon nanotubes into electronic, optical and consumer products will inevitably lead to the presence of these anthropogenic compounds in the environment. To date, there have been few studies isolating these materials from environmental matrices. Here we report a method commonly used to quantify black carbon (BC) in soils, the benzene polycarboxylic acid (BPCA) method, for measurement of two types of single walled carbon nanotubes (SWCNTs), two types of fullerenes and two forms of soot. The distribution of BC products (BPCAs) from the high pressure and high temperature oxidation illustrates the condensed nature of these compounds because they form predominantly fully substituted mellitic acid (B6CA). The conversion of carbon nanoparticles to BPCAs was highest for fullerenes (average of 23.2 ± 4.0% C recovered for both C60 and C70) and lowest for non-functionalized SWCNTs (0.5 ± 0.1% C). The recovery of SWCNTs was 10 times higher when processed through a cation-exchange column, indicating the presence of metals in SWCNTs compromises the oxidation chemistry. While mixtures of SWCNTs, soot and sediment revealed small losses of black carbon during sample processing, the method is suitable for quantifying total BC. The BPCA distribution of mixtures did not agree with theoretical mixtures using model polyaromatic hydrocarbons, suggesting the presence of a matrix effect. Future work is required to quantify different types of black carbon within the same sample.

  7. 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.

  8. Antibiotic Resistance and the Frequency of Extended-Spectrum B-Lactamase in Acinetobacter baumannii Isolated from Clinical Samples through Phenotypic Methods

    Directory of Open Access Journals (Sweden)

    Somayeh Vafaei

    2013-07-01

    Full Text Available AbstractBackground and objectives: Nowadays Acinetobacter baumannii is as one of the problematic opportunistic pathogens, especially in intensive care because of the incidence of drug-resistant strains in the world. The purpose of current study was to define the antibiotic susceptibility patterns and detect the prevalence of producing strains of extended-spectrum β-lactamase (ESBL in A. baumannii isolates which had been isolated from clinical samples with combined disk test.Materials and methods: This study was conducted in 3 major hospitals in Tehran on 500 clinical samples during 6 months. After identification of isolates in species level using cultural and biochemical methods, in order to determine sensitivity of 100 isolates of A. baumannii to 11 antibiotics, the susceptibility tests were carried out according to CLSI guidelines using disk diffusion method. Also MIC (Minimum inhibitory concentrations was determined for cefepime and ceftazidime, and finally to identify of producing strains of ESBL was applied phenotypic method of combined disk. Results: In this survey, 100 A. baumannii strains, 30 A. lwoffii strains and other Acinetobacter species were isolated from patients. The majority of isolates were from blood specimens. Isolates of A.baumannii revealed the highest resistance to cefepime, ceftriaxone, amikacin, imipenem, piperacillin - tazobactam, meropenem, gentamicin, tobramycina and tetracycline, respectively. Ampicillin - sulbactam and polymyxin B considered as effective drugs in this study. Multi-drug resistance in these strains was 70%. The Total isolates studied the Minimum inhibitory concentrations of ceftazidime in 84% samples was MIC ≥ 128 μg/ml and Minimum inhibitory concentrations of cefepime in 91% samples was MIC≥ 128 μg/ml. According to the results of combined disk test, 20% of total samples were demonstrated to be ESBL positive.Conclusion: Regarding to produce of ESBL in this bacterium and possibility of

  9. A fast method to estimate speciation parameters in a model of isolation with an initial period of gene flow and to test alternative evolutionary scenarios

    OpenAIRE

    Wilkinson-Herbots, Hilde

    2015-01-01

    We consider a model of "isolation with an initial period of migration" (IIM), where an ancestral population instantaneously split into two descendant populations which exchanged migrants symmetrically at a constant rate for a period of time but which are now completely isolated from each other. A method of Maximum Likelihood estimation of the parameters of the model is implemented, for data consisting of the number of nucleotide differences between two DNA sequences at each of a large number ...

  10. Identification of a 12-gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics.

    Science.gov (United States)

    Brown, Daren W; Lee, Seung-Ho; Kim, Lee-Han; Ryu, Jae-Gee; Lee, Soohyung; Seo, Yunhee; Kim, Young Ho; Busman, Mark; Yun, Sung-Hwan; Proctor, Robert H; Lee, Theresa

    2015-03-01

    In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production. PMID:25372119

  11. Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach

    Directory of Open Access Journals (Sweden)

    Garvin Alex M

    2012-12-01

    Full Text Available Abstract 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.

  12. The isolated chicken eye test as a suitable in vitro method for determining the eye irritation potential of household cleaning products

    NARCIS (Netherlands)

    Schutte, K.; Prinsen, M.K.; McNamee, P.M.; Roggeband, R.

    2009-01-01

    Eye irritation is an important endpoint in the safety evaluation of consumer products and their ingredients. Several in vitro methods have been developed and are used by different industry sectors to assess eye irritation. One such in vitro method in use for some time already is the isolated chicken

  13. Serological studies on British isolates of the Sejroe serogroup of leptospira. II. An evaluation of the factor analysis method of identifying leptospires using strains belonging to the Sejroe serogroup.

    OpenAIRE

    Little, T. W.; Stevens, A. E.; Hathaway, S. C.

    1987-01-01

    Twelve British isolates of leptospira belonging to the Sejroe serogroup were examined using a series of six factor sera prepared by a number of different absorption methods. Ten of the isolates were identified as Leptospira interrogans serovar hardjo and two as L. interrogans serovar saxkoebing. These isolates had previously been identified using the cross agglutination absorption method.

  14. Fluorescent profiling of modular biosynthetic enzymes by complementary metabolic and activity based probes.

    Science.gov (United States)

    Meier, Jordan L; Mercer, Andrew C; Burkart, Michael D

    2008-04-23

    The study of the enzymes responsible for natural product biosynthesis has proven a valuable source of new enzymatic activities and been applied to a number of biotechnology applications. Protein profiling could prove highly complementary to genetics based approaches by allowing us to understand the activity, transcriptional control, and post-translational modification of these enzymes in their native and dynamic proteomic environments. Here we present a method for the fluorescent profiling of PKS, NRPS, and FAS multidomain modular synthases in their whole proteomes using complementary metabolic and activity based probes. After first examining the reactivity of these activity based probes with a variety of purified recombinant PKS, NRPS, and FAS enzymes in vitro, we apply this duel labeling strategy to the analysis of modular synthases in a human breast cancer cell line and two strains of the natural product producer Bacillus subtilis. Collectively, these studies demonstrate that complementary protein profiling approaches can prove highly useful in the identification and assignment of inhibitor specificity and domain structure of these modular biosynthetic enzymes. PMID:18376827

  15. Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor.

    Science.gov (United States)

    Qu, Xudong; Jiang, Nan; Xu, Fei; Shao, Lei; Tang, Gongli; Wilkinson, Barrie; Liu, Wen

    2011-03-01

    Sanglifehrin A (SFA), a potent cyclophilin inhibitor produced by Streptomyces flaveolus DSM 9954, bears a unique [5.5] spirolactam moiety conjugated with a 22-membered, highly functionalized macrolide through a linear carbon chain. SFA displays a diverse range of biological activities and offers significant therapeutic potential. However, the structural complexity of SFA poses a tremendous challenge for new analogue development via chemical synthesis. Based on a rational prediction of its biosynthetic origin, herein we report the cloning, sequencing and characterization of the gene cluster responsible for SFA biosynthesis. Analysis of the 92 776 bp contiguous DNA region reveals a mixed polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) pathway which includes a variety of unique features for unusual PKS and NRPS building block formation. Our findings suggest that SFA biosynthesis requires a crotonyl-CoA reductase/carboxylase (CCR) for generation of the putative unusual PKS starter unit (2R)-2-ethylmalonamyl-CoA, an iterative type I PKS for the putative atypical extender unit (2S)-2-(2-oxo-butyl)malonyl-CoA and a phenylalanine hydroxylase for the NRPS extender unit (2S)-m-tyrosine. A spontaneous ketalization of significant note, may trigger spirolactam formation in a stereo-selective manner. This study provides a framework for the application of combinatorial biosynthesis methods in order to expand the structural diversity of SFA. PMID:21416665

  16. Multigrid methods and high order finite difference for flow in transition - Effects of isolated and distributed roughness elements

    Science.gov (United States)

    Liu, C.; Liu, Z.

    1993-01-01

    The high order finite difference and multigrid methods have been successfully applied to direct numerical simulation (DNS) for flow transition in 3D channels and 3D boundary layers with 2D and 3D isolated and distributed roughness in a curvilinear coordinate system. A fourth-order finite difference technique on stretched and staggered grids, a fully-implicit time marching scheme, a semicoarsening multigrid method associated with line distributive relaxation scheme, and a new treatment of the outflow boundary condition, which needs only a very short buffer domain to damp all wave reflection, are developed. These approaches make the multigrid DNS code very accurate and efficient. This makes us not only able to do spatial DNS for the 3D channel and flat plate at low computational costs, but also able to do spatial DNS for transition in the 3D boundary layer with 3D single and multiple roughness elements. Numerical results show good agreement with the linear stability theory, the secondary instability theory, and a number of laboratory experiments.

  17. A New Method for Start-up of Isolated Boost Converters Using Magnetic- and Winding-Integration

    DEFF Research Database (Denmark)

    Lindberg-Poulsen, Kristian; Ouyang, Ziwei; Sen, Gökhan;

    2012-01-01

    A new solution to the start-up and low output voltage operation of isolated boost family converters is presented. By the use of integrated magnetics and winding integration, the transformer secondary winding is re-used during start-up as a flyback winding coupled to the boost inductor. The...... extended to other isolated boost family topologies. The principle of operation is demonstrated with a 800W isolated boost prototype, and a 1600W primary parallel series secondary isolated boost converter. Efficiency measurements of both prototypes are presented, including measurements during both start-up...

  18. 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.

  19. Isolation of rat hepatic stellate cells by nonperfusion method and identification of isolated cells%非灌流法分离大鼠肝星形细胞及其鉴定

    Institute of Scientific and Technical Information of China (English)

    刘朋飞; 周余来; 冯业童; 吴昊昱; 刘迪; 董超; 吴璇; 石毅

    2012-01-01

    目的 采用非灌流法分离大鼠肝星形细胞(Hepatic stellate cell,HSC),并进行鉴定.方法 采用非灌流法结合酶消化法分离大鼠肝脏细胞,密度梯度离心进一步分离HSC,油红染色检测HSC胞质中的脂滴,免疫组化法检测细胞中α-平滑肌肌动蛋白(α-Smooth muscle actin,α-SMA)、结蛋白(Desmin)及神经胶质酸性蛋白(Glial fibrillary acidic protein,GFAP)的表达.结果 非灌流法结合酶消化法可成功分离大鼠HSC;密度梯度离心纯化的HSC经油红染色,细胞核周围可见红色脂滴;该细胞中α-SMA、结蛋白及GFAP的免疫组化染色结果均呈阳性,细胞着色率可达90%以上.结论 成功建立了大鼠HSC的非灌流分离模式,所获得的HSC纯度较高,该方法稳定简便,具有一定的推广应用价值.%Objective To isolate rat hepatic stellate cells (HSCs) by nonperfusion method and identify the isolated cells. Methods Rat liver cells were isolated by nonperfusion method combined with enzyme digestion method, from which HSCs were further isolated by density gradient centrifugation. The lipid droplets in cytoplasm of HSCs were determined by oil red staining, while the expressions of a-smooth muscle actin (a-SMA), desmin and neuroglia acid protein (GFAP) by immunohistochemical staining. Results Rat HSCs were successfully isolated by nonperfusion method combined with enzyme digestion method. After oil red staining, red lipid droplets were found around the nucleus in HSCs purified by density gradient centrifugation. All the positive rates of a-SMA, desmin and GFAP were more than 90% in immunohistochemical staining. Conclusion The nonperfusion isolating mode of rat HSC was established successfully, by which highly purified HSCs were obtained. The method was stable, simple, and worthy of popularization.

  20. 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)

  1. Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.

    Science.gov (United States)

    Smith, J Robert; Pfeifer, Kyle; Petry, Florian; Powell, Natalie; Delzeit, Jennifer; Weiss, Mark L

    2016-01-01

    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. PMID:26966439

  2. Isolation of Chlorella vulgaris and Its DNA Extraction Methods%小球藻的分离及其DNA提取方法的研究

    Institute of Scientific and Technical Information of China (English)

    王恒强; 孔庆军; 任雪艳; 占东霞; 张海黎

    2008-01-01

    [Objective] The aim of this study is to isolate Chlorella vulgaris (chlorella) and extract its genomic DNA. [Method] Both the dilution method and drip method were employed to isolate chlorella from lake water samples; the conditions for culturing chlorella were optimized and its genomic DNA was extracted by improved CTAB method and SDS method. [Result] The proper conditions for chlorella culture were as following: temperature 20-25 ℃, illumination 4.39-5.86 W/m2 and rotational speed 100-150r/min; improved CTAB method was suitable for extracting genomic DNA from chlorella. [Conclusion] The study is helpful to study the chlorella at molecular level and promote the exploitation and utilization of chlorella resources.

  3. Localization and interactions between Arabidopsis auxin biosynthetic enzymes in the TAA/YUC-dependent pathway.

    Science.gov (United States)

    Kriechbaumer, Verena; Botchway, Stanley W; Hawes, Chris

    2016-07-01

    The growth regulator auxin is involved in all key developmental processes in plants. A complex network of a multiplicity of potential biosynthetic pathways as well as transport, signalling plus conjugation and deconjugation lead to a complex and multifaceted system system for auxin function. This raises the question how such a system can be effectively organized and controlled. Here we report that a subset of auxin biosynthetic enzymes in the TAA/YUC route of auxin biosynthesis is localized to the endoplasmic reticulum (ER). ER microsomal fractions also contain a significant percentage of auxin biosynthetic activity. This could point toward a model of auxin function using ER membrane location and subcellular compartmentation for supplementary layers of regulation. Additionally we show specific protein-protein interactions between some of the enzymes in the TAA/YUC route of auxin biosynthesis. PMID:27208541

  4. AN EVALUATION OF VARIOUS METHODS FOR THE DETECTION OF METALLO-Β-LACTAMASE IN CLINICAL ISOLATES OF PSEUDOMONAS AERUGINOSA IN A TEACHING HOSPITAL OF RURAL GUJARAT, INDIA

    Directory of Open Access Journals (Sweden)

    Priyanka

    2014-09-01

    Full Text Available BACKGROUND: Metallo-β-Lactamase (MBL mediated resistance to various antibiotics is an emerging threat in Pseudomonas aeruginosa isolates. The present study was undertaken to detect MBL producing isolates of Pseudomonas aeruginosa from various clinical specimen by four different phenotypic methods. OBJECTIVE: To evaluate the various methods for the detection of Metallo-β-Lactamase in clinical isolates of Pseudomonas aeruginosa in a teaching hospital of rural Gujarat – India. STUDY SETTING: Department of Microbiology, P.S. Medical College & Shree Krishna Hospital, Karamsad, Gujarat. STUDY DESIGN: Prospective observational. MATERIALS AND METHODS: Total 50 isolates of Pseudomonas aeruginosa isolated & identified from different clinical specimens as per the standard guidelines. Screening for MBL production was done by Imipenem-EDTA combined disc test, Imipenem-EDTA double disc synergy test (DDST, EDTA disc potentiation using four cephalosporins, MBL E-test. RESULT: Of 50 imipenem sensitive or resistant isolates, 24 were MBL positive, Of which 12(24% were MBL positive by Imipenem-EDTA combined disc test and Imipenem-EDTA double disc synergy test (DDST, 2(4% were MBL positive by EDTA disc potentiation using four cephalosporins and 19(63.33% were MBL positive by MBL E-test method. CONCLUSION: Imipenem-EDTA combined disc test and Imipenem-EDTA double disc synergy test (DDST are equally effective for MBL detection while MBL E-test is more effective for MBL detection but given the cost-constraints, combined disc test and DDST can be used as a convenient screening method in the clinical microbiology laboratory.

  5. Draft Genome Sequence of Insecticidal Streptomyces sp. Strain PCS3-D2, Isolated from Mangrove Soil in Philippines

    OpenAIRE

    Bayot-Custodio, Aileen N.; Alcantara, Edwin P.; Zulaybar, Teofila O.

    2014-01-01

    A draft genome sequence of a Streptomyces sp. isolated from mangrove soil in Cebu, Philippines, is described here. This isolate produced compounds with contact insecticidal activity against important corn pests. The genome contains 7,479,793 bp (in 27 scaffolds), 6,297 predicted genes, and 29 secondary metabolite biosynthetic gene clusters.

  6. Application of Different Drying Methods on β-Glucan Isolated from Spent Brewer’s Yeast Using Alkaline Procedure

    Directory of Open Access Journals (Sweden)

    Vesna Zechner-Krpan

    2014-02-01

    Full Text Available Normal 0 false false false 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-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} Water-insoluble (particulate β-glucan was isolated from the cell walls of spent brewer’s yeast using a single-step alkaline treatment. Sonication was successfully used to stabilize water suspensions of b-glucan. Three different drying methods were used: air-drying, lyophilization and spray-drying. Air-drying and lyophilization caused changes of β-glucan microstructure and particle agglomeration. Sonication combined with spray-drying resulted in minimal β-glucan structural changes and negligible formation of agglomerates. Re-aggregation of spray-dried β-glucan particles was minimal even after re-suspending in water.

  7. Molecular characterization ofAcidithiobacillus ferrooxidans strains isolated from different environments by three PCR-based methods

    Institute of Scientific and Technical Information of China (English)

    吴学玲; 刘莉莉; 张真真; 刘新星; 邓凡凡

    2015-01-01

    PCR-based DNA fingerprinting, REP-PCR (repetitive element PCR), RAPD (randomly amplified polymorphic DNA) and 16S rDNA sequence analyses were used to characterize 23Acidithiobacillus ferrooxidansstrains isolated from different environments. (GTG)5 and BOXA1R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles fromA. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains ofA. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using (GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16S rDNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a newAcidithiobacillus namedAcidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.

  8. 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%. PMID:27374526

  9. 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

    2010-06-22

    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

  10. Optimization and Isolation of Dermatophytes from Clinical Samples and In Vitro Antifungal Susceptibility Testing By Disc Diffusion Method

    Directory of Open Access Journals (Sweden)

    Amodkumar Yadav

    2013-06-01

    Full Text Available India is a large subcontinent with remarkably varied topography, situated within the tropical and subtropical belts of the world. In the study patients with Tinea infections were examined clinically by dermatologist. Isolation, confirmatory test were done as per the standard procedure, and Antifungal Susceptibility test was done by Disc diffusion method.Other conditions such as seborrheic dermatitis, psoriasis, alopecia areata, folliculitis and pseudopelade may mimic ringworm of head and other tinea must be identified. A total of sixty six patients of dermatophytosis were studied. Males were predominantly affected 51 (77% cases as compared to females15 (23% cases. Male to female ratio was 3.4:1. Most common age group affected was 21-30 years with 20 cases (23%. Least common age group affected was 61-70 years with 3 cases (4%.Tinea corporis was more common in the age group 21-30 years with 13 cases (37.14% and in males with 29 cases (82.85% than females with 6 cases (17.15%.Tinea unguium was more common in the age group of 31-40 years with 6 cases (37.5% and in males with 10 cases (62.5% than females with 6 cases (37.5%.Tinea cruris was more common in the age group 51-60 years with 2 cases (40% and was more common in males with 5 cases (100%.In tinea pedis, one case was seen in the age group of 11-20 years and the other in the age group of 41-50 and 51-60 years, and was more common in males with 3 cases (100%.Tinea barbae was more common in the age group 21-30 years with 2 cases (66.66% .Tinea capitis was more common in the age group of 31-40 years with 2 cases (66.66% and was more common in females with 3 cases (100%. Tinea manuum was more common in the age group of 31-40 years and in males with 1 case (100%. In males, commonest infection was T. corporis while in female commonest infection was T.corporis.rate of direct microscopy and culture (78.79%. About 89.47% of the dermatophytes grew faster in DTM with compare to SDA, so the growth rate of

  11. 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)

    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

  12. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae.

    Science.gov (United States)

    He, Yanxia; Guo, Xianguang; Xiang, Shifei; Li, Jiao; Li, Xiaoqin; Xiang, Hui; He, Jinlei; Chen, Dali; Chen, Jianping

    2016-07-01

    The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae. PMID:27147066

  13. 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

  14. Comparative Analysis of the Biosynthetic Gene Clusters and Pathways for Three Structurally Related Antitumor Antibiotics Bleomycin, Tallysomycin and Zorbamycin†

    OpenAIRE

    Galm, Ute; Wendt-Pienkowski, Evelyn; Wang, Liyan; Huang, Sheng-Xiong; Unsin, Claudia; Tao, Meifeng; Coughlin, Jane M.; Shen, Ben

    2011-01-01

    The biosynthetic gene clusters for the glycopeptide antitumor antibiotics bleomycin (BLM), tallysomycin (TLM), and zorbamycin (ZBM) have been recently cloned and characterized from Streptomyces verticillus ATCC15003, Streptoalloteichus hindustanus E465-94 ATCC31158, and Streptomyces flavoviridis ATCC21892, respectively. The striking similarities and differences among the biosynthetic gene clusters for the three structurally related glycopeptide antitumor antibiotics prompted us to compare and...

  15. Variability in mycotoxin biosynthetic genes and gene clusters in Fusarium and its implications for mycotoxin contamination of crops

    Science.gov (United States)

    The Fusarium metabolites fumonisins and trichothecenes are among the mycotoxins of greatest concern to food and feed safety worldwide. As with other fungal secondary metabolites, mycotoxin biosynthetic genes are often located adjacent to one another in gene clusters. Thus, fumonisin biosynthetic gen...

  16. Effects of overexpressing individual lignin biosynthetic enzymes on feeding and growth of corn earworms and fall armyworms

    Science.gov (United States)

    Lignin is an important insect resistance component of plants. Enhancing or disrupting the lignin biosynthetic pathway for different bioenergy uses may alter pest resistance. The lignin biosynthetic pathway is complex, and a number of pathway compounds are also involved in the biosynthesis of simpler...

  17. 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...

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

    OpenAIRE

    Woo, JungReem; Sharma, Shivani; Gimzewski, James

    2016-01-01

    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 d...

  19. 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 ...

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

    Science.gov (United States)

    Bach, Søren Spanner; King, Brian Christopher; Zhan, Xin; Simonsen, Henrik Toft; Hamberger, Björn

    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 streamlining of large scale Agrobacterium infiltration and upregulation of the upstream pathways, transient in planta heterologous expression quickly reaches limitations when used for production of terpenoids. Stable integration of transgenes into the nuclear genome of the moss Physcomitrella patens has already been widely recognized as a viable alternative for industrial-scale production of biopharmaceuticals. For expression of terpenoid biosynthetic genes, and reconstruction of heterologous pathways, Physcomitrella has unique attributes that makes it a very promising biotechnological host. These features include a high native tolerance to terpenoids, a simple endogenous terpenoid profile, convenient genome editing using homologous recombination, and cultivation techniques that allow up-scaling from single cells in microtiter plates to industrial photo-bioreactors. Beyond its use for functional characterization of terpenoid biosynthetic genes, engineered Physcomitrella can be a green biotechnological platform for production of terpenoids. Here, we describe two complementary and simple procedures for stable nuclear transformation of Physcomitrella with terpenoid biosynthetic genes, selection and cultivation of transgenic lines, and metabolite analysis of terpenoids produced in transgenic moss lines. We also provide tools for metabolic engineering through genome editing using homologous recombination. PMID:24777804

  1. SELECTIVE SEPARATION OF BIOSYNTHETIC PRODUCTS BY PERTRACTION - CHALLENGE FOR THE “WHITE BIOTECHNOLOGY”

    OpenAIRE

    Dan Cascaval; Anca-Irina Galaction

    2010-01-01

    This review presents our original results on selective separation of some biosynthetic products (antibiotics, carboxylic acids, amino acids) by free or facilitated pertraction (extraction and transport through liquid membranes). Selecting the optimum conditions, for all studied cases these pertraction technique simplify the technologies applied at industrial scale for separation and purification, allows to reaching high selectivity and reducing the overall cost of the products.

  2. Sugars as the optimal biosynthetic carbon substrate of aqueous life throughout the universe

    Science.gov (United States)

    Weber, A. L.

    2000-01-01

    Our previous analysis of the energetics of metabolism showed that both the biosynthesis of amino acids and lipids from sugars, and the fermentation of organic substrates, were energetically driven by electron transfer reactions resulting in carbon redox disproportionation (Weber, 1997). Redox disproportionation--the spontaneous (energetically favorable) direction of carbon group transformation in biosynthesis--is brought about and driven by the energetically downhill transfer of electron pairs from more oxidized carbon groups (with lower half-cell reduction potentials) to more reduced carbon groups (with higher half-cell reduction potentials). In this report, we compare the redox and kinetic properties of carbon groups in order to evaluate the relative biosynthetic capability of organic substrates, and to identify the optimal biosubstrate. This analysis revealed that sugars (monocarbonyl alditols) are the optimal biosynthetic substrate because they contain the maximum number of biosynthetically useful high energy electrons/carbon atom while still containing a single carbonyl group needed to kinetically facilitate their conversion to useful biosynthetic intermediates. This conclusion applies to aqueous life throughout the Universe because it is based on invariant aqueous carbon chemistry--primarily, the universal reduction potentials of carbon groups.

  3. Multi-responsive physical gels formed by a biosynthetic asymmetric triblock protein polymer and a polyanion

    NARCIS (Netherlands)

    Pham, T.H.T.; Wang, J.; Werten, M.W.T.; Snijkers, F.; Wolf, de F.A.; Cohen Stuart, M.A.; Gucht, van der J.

    2013-01-01

    We report the design, production and characterization of a biosynthetic asymmetric triblock copolymer which consists of one collagen-like and one cationic block spaced by a hydrophilic random coiled block. The polymer associates into micelles when a polyanion is added due to the electrostatic intera

  4. Quantification of trichothecene biosynthetic genes during the growth cycle of Fusarium sporotrichioides in culture

    Science.gov (United States)

    Trichothecene mycotoxins are secondary metabolites produced by several species of phytopathogenic fungi, and are potent inhibitors of protein biosynthesis. The genes involved in the biosynthetic pathway of T-2 toxin in Fusarium sporotrichioides have been characterized and are located in four identi...

  5. Binding of insulin to rat pancreatic islets: comparison between pancreatic human insulin and biosynthetic human insulin

    Energy Technology Data Exchange (ETDEWEB)

    Verspohl, E.J.; Ammon, H.P.

    Human pancreatic insulin, biosynthetic human insulin (BHI), and pork insulin were compared in terms of their binding characteristics to insulin receptors on rat pancreatic islets. There was no difference in binding or on biologic effect, i.e., ability to inhibit insulin secretion.

  6. HPLC-SPE-NMR for combinatorial biosynthetic investigations – Expanding the landscape of diterpene structural diversity

    DEFF Research Database (Denmark)

    Kongstad, Kenneth Thermann; Andersen-Ranberg, Johan; Hamberger, Björn Robert;

    In this work, the analytical technique, HPLC-HRMS-SPE-NMR was used for the first time in combination with combinatorial biosynthetic investigations in N. benthamiana. This efficient setup allowed for identification of several diterpene synthase (diTPS) combinations responsible for stereospecific...

  7. 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

  8. The Biosynthetic Pathways of Tanshinones and Phenolic Acids in Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Xiao-Hui Ma

    2015-09-01

    Full Text Available Secondary metabolites from plants play key roles in human medicine and chemical industries. Due to limited accumulation of secondary metabolites in plants and their important roles, characterization of key enzymes involved in biosynthetic pathway will enable metabolic engineering or synthetic biology to improve or produce the compounds in plants or microorganisms, which provides an alternative for production of these valuable compounds. Salvia miltiorrhiza, containing tanshinones and phenolic acids as its active compounds, has been widely used for the treatment of cardiovascular and cerebrovascular diseases. The biosynthetic analysis of secondary metabolites in S. miltiorrhiza has made great progress due to the successful genetic transformation system, simplified hairy roots system, and high-throughput sequencing. The cloned genes in S. miltiorrhiza had provided references for functional characterization of the post-modification steps involved in biosynthesis of tanshinones and phenolic acids, and further utilization of these steps in metabolic engineering. The strategies used in these studies could provide solid foundation for elucidation of biosynthetic pathways of diterpenoids and phenolic acids in other species. The present review systematically summarizes recent advances in biosynthetic pathway analysis of tanshinones and phenolic acids as well as synthetic biology and metabolic engineering applications of the rate-limiting genes involved in the secondary metabolism in S. miltiorrhiza.

  9. A comparison of six total rna isolation methods for diagnosis of gysvd-1 (grapevine yellow speckle viroid-1) on vitis vinifera L. leaves

    International Nuclear Information System (INIS)

    Isolation of high quality RNA from plant tissues is one of the most critical steps for the successful application of diagnostic tests such as reverse transcription polymerase chain reaction (RT-PCR), northern blotting, microarray hybridization. The presence of inhibitors such as secondary metabolites, phenolic compounds and RNAses can cause inaccurate and undesirable results. Grapevine is rich in a wide range of metabolites which interfere with RNA isolation. From this point of view, we researched six different total RNA extraction methods on leaves of Vitisvinifera L. to find the best one that contribute the purity and high quality. The methods tested are silica-capture, modified silica-capture, commercial kit, the new combined, lithium chloride and citric buffer. RNA quality was analyzed spectrophotometrically by nanodrop, agarose gel electrophoresis and RT-PCR. As a result of all, it is clear that the most suitable TNA isolation protocol is the new combined method which experienced and named firstly by us, in terms of RNA purity, concentration, less time consuming of isolation step and achievement on detection of GYSVd-1. (author)

  10. 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)

    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)

  11. Nanostructuring biosynthetic hydrogels for tissue engineering: a cellular and structural analysis.

    Science.gov (United States)

    Frisman, Ilya; Seliktar, Dror; Bianco-Peled, Havazelet

    2012-01-01

    The nanostructuring of hydrogel scaffolds used in tissue engineering provides the ability to control cellular fate and tissue morphogenesis through cell-matrix interactions. Here we describe a method to provide nanostructure to a biosynthetic hydrogel scaffold made from crosslinked poly(ethylene glycol)-fibrinogen conjugates (PEG-fibrinogen), by modifying them with the block-copolymer Pluronic® F127. The copolymeric additive self-assembled into micelles at certain concentrations and temperatures, thereby creating nanostructures within the crosslinked hydrogel. Small-angle X-ray scattering (SAXS) and transmission electron microscopy at cryogenic temperature were used to detect Pluronic® F127 micelles embedded within the crosslinked PEG-fibrinogen hydrogels. The density and order of the micelles within the hydrogel matrix increased as the relative Pluronic® F127 concentration was raised. The transient stability of the micelles within the hydrogel network was analyzed using time-dependent swelling and Pluronic® F127 release measurements. These characterizations revealed that most of the Pluronic® F127 molecules diffuse out of the hydrogels after 4 days in aqueous buffer and SAXS analysis confirmed a significant change in the structure and interactions of the micelles during this time. Cell culture experiments evaluating the three-dimensional fibroblast morphology within the matrix indicated a strong correlation between cell spreading and the hydrogel's characteristic mesh size. The present research thereby provides a more quantitative understanding of how structural features in an encapsulating hydrogel environment can affect cell morphogenesis towards tissue regeneration. PMID:21855662

  12. Bioenergetic coupling between membrane transport systems and biosynthetic pathways essential for cell cycle progression

    International Nuclear Information System (INIS)

    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 G1 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 3H-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, 3H-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

  13. 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.

  14. Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+ MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

    Directory of Open Access Journals (Sweden)

    Zsuzsa Kreizinger

    Full Text Available Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+ MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

  15. Design method of vertical isolation system for fast reactor components and trial manufacture of disk-springs

    International Nuclear Information System (INIS)

    Common deck formula vertical isolation system for fast reactor components and seismic isolation elements are investigated. The following results are obtained: 1) the design standards (JIS, DIN and SAE) for disk-spring were studied. JIS and SAE are made on the basis of DIN. The disk-spring for 1500,000 kWe sodium cooled loop reactor was designed; SUP10 materials, φ1000 mm external diameter, 27 mm thickness of plate, 5 x 14 parallel number x series number, 275 tonf supporting weight/unit and 28 number of disk-spring unit. Damper and horizontal supporting device for common deck are designed. Arrangement of disk-spring and deck are designed. 2) Real size disk-spring was test manufactured. It made possible to obtain data for verification of produce. Guideline for design of common deck formula vertical isolation system for fast reactor components was made by these results. (S.Y.)

  16. A simple, fast, and inexpensive CTAB-PVP-silica based method for genomic DNA isolation from single, small insect larvae and pupae.

    Science.gov (United States)

    Huanca-Mamani, W; Rivera-Cabello, D; Maita-Maita, J

    2015-01-01

    In this study, we report a modified CTAB-PVP method combined with silicon dioxide (silica) treatment for the extraction of high quality genomic DNA from a single larva or pupa. This method efficiently obtains DNA from small specimens, which is difficult and challenging because of the small amount of starting tissue. Maceration with liquid nitrogen, phenol treatment, and the ethanol precipitation step are eliminated using this methodology. The A260/A280 absorbance ratios of the isolated DNA were approximately 1.8, suggesting that the DNA is pure and can be used for further molecular analysis. The quality of the isolated DNA permits molecular applications and represents a fast, cheap, and effective alternative method for laboratories with low budgets. PMID:26214482

  17. Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

    Science.gov (United States)

    Francois, Patrice; Huyghe, Antoine; Charbonnier, Yvan; Bento, Manuela; Herzig, Sébastien; Topolski, Ivan; Fleury, Bénédicte; Lew, Daniel; Vaudaux, Pierre; Harbarth, Stephan; van Leeuwen, Willem; van Belkum, Alex; Blanc, Dominique S.; Pittet, Didier; Schrenzel, Jacques

    2005-01-01

    Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time. PMID:16000459

  18. Evaluation of the Antimicrobial Activity of the K9CATH Peptide (38 Amino Acids Against a Mastitis Isolated Strain of Staphylococcus aureus by the Resazurin microtiter Method

    Directory of Open Access Journals (Sweden)

    Albero Barreras-Serrano

    2014-04-01

    Full Text Available The antimicrobial activity of the synthetic peptide K9CATH was determined by the Resazurin microtitre Method (RMM against a strain of S. aureus isolated from a case of mastitis. To the antibiogram this bacteria strain showed to be resistant to Ampicillin, Erythromycin, Cefeprime, Dicloxaciline and Penicillin (10 U, while the MIC obtained for the K9CATH was 5.66 &mug/mL. Unlike the reference broth method, visual reading for MIC determination with the RMM showed to be easier, rapid, inexpensive and more sensitive for antimicrobial peptide screening, based in a color change from blue (not growth to pink (growth. This is the first time that the resazurin method is used to determine the MIC of the 38 aa´s K9CATH peptide against a mastitic isolate of S. aureus.

  19. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10.

    Science.gov (United States)

    Liu, Shui-Ping; Yuan, Peng-Hui; Wang, Yue-Yue; Liu, Xiao-Fang; Zhou, Zhen-Xing; Bu, Qing-ting; Yu, Pin; Jiang, Hui; Li, Yong-Quan

    2015-04-01

    The polyene antibiotic natamycin is widely used as an antifungal agent in both human therapy and the food industry. Here we obtained four natamycin analogs with high titers, including two new compounds, by engineering of six post-polyketide synthase (PKS) tailoring enzyme encoding genes in a natamycin industrial producing strain, Streptomyces chattanoogensis L10. Precise analysis of S. chattanoogensis L10 culture identified natamycin and two natamycin analogs, 4,5-deepoxy-natamycin and 4,5-deepoxy-natamycinolide. The scnD deletion mutant of S. chattanoogensis L10 did not produce natamycin but increased the titer of 4,5-deepoxy-natamycin. Inactivation of each of scnK, scnC, and scnJ in S. chattanoogensis L10 abolished natamycin production and accumulated 4,5-deepoxy-natamycinolide. Deletion of scnG in S. chattanoogensis L10 resulted in production of two new compounds, 4,5-deepoxy-12-decarboxyl-12-methyl-natamycin and its dehydration product without natamycin production. Inactivation of the ScnG-associated ferredoxin ScnF resulted in impaired production of natamycin. Bioassay of these natamycin analogs showed that three natamycin analogs remained antifungal activities. We found that homologous glycosyltransferases genes including amphDI and nysDI can partly complement the ΔscnK mutant. Our results here also support that ScnG, ScnK, and ScnD catalyze carboxylation, glycosylation, and epoxidation in turn in the natamycin biosynthetic pathway. Thus this paper provided a method to generate natamycin analogs and shed light on the natamycin biosynthetic pathway. PMID:25801968

  20. Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

    Science.gov (United States)

    Ovchinnikova, Tatyana V; Shenkarev, Zakhar O; Yakimenko, Zoya A; Svishcheva, Natalia V; Tagaev, Andrey A; Skladnev, Dmitry A; Arseniev, Alexander S

    2003-01-01

    Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution. PMID:14658801

  1. Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq

    Directory of Open Access Journals (Sweden)

    Xiasheng Zheng

    2014-04-01

    Full Text Available Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield. According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins.

  2. 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.

  3. Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

    Directory of Open Access Journals (Sweden)

    Honghao Chen

    2013-12-01

    Full Text Available Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic. Based on previous research, these effects are mediated by a number of active ingredients, especially glycyrrhizic acid (GA. In the present study, a gene encoding β-amyrin synthase (β-AS involved in GA biosynthesis in G. uralensis has been cloned and expressed in Saccharomyces cerevisiae. The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene into β-amyrin. In fact the β-AS gene is particularly important in the GA biosynthetic pathway in G. uralensis. The complete sequence of the enzyme was determined and a phylogenetic tree based on the β-AS gene of G. uralensis and 20 other species was created. This showed that Glycyrrhiza glabra had the closest kinship with G. uralensis. The results of this work will be useful in determining how to improve the efficacy of G. uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

  4. Kinetic model of metabolic network for xiamenmycin biosynthetic optimisation.

    Science.gov (United States)

    Xu, Min-juan; Chen, Yong-cong; Xu, Jun; Ao, Ping; Zhu, Xiao-mei

    2016-02-01

    Xiamenmycins, a series of prenylated benzopyran compounds with anti-fibrotic bioactivities, were isolated from a mangrove-derived Streptomyces xiamenensis. To fulfil the requirements of pharmaceutical investigations, a high production of xiamenmycin is needed. In this study, the authors present a kinetic metabolic model to evaluate fluxes in an engineered Streptomyces lividans with xiamenmycin-oriented genetic modification based on generic enzymatic rate equations and stability constraints. Lyapunov function was used for a viability optimisation. From their kinetic model, the flux distributions for the engineered S. lividans fed on glucose and glycerol as carbon sources were calculated. They found that if the bacterium can utilise glucose simultaneously with glycerol, xiamenmycin production can be enhanced by 40% theoretically, while maintaining the same growth rate. Glycerol may increase the flux for phosphoenolpyruvate synthesis without interfering citric acid cycle. They therefore believe this study demonstrates a possible new direction for bioengineering of S. lividans. PMID:26816395

  5. Whole-Genome Sequence of Bacillus sp. SDLI1, Isolated from the Social Bee Scaptotrigona depilis

    Science.gov (United States)

    Paludo, Camila R.; Silva-Junior, Eduardo A.; Pishchany, Gleb; Currie, Cameron R.; Nascimento, Fábio S.; Kolter, Roberto G.

    2016-01-01

    We announce the complete genome sequence of Bacillus sp. strain SDLI1, isolated from larval gut of the stingless bee Scaptotrigona depilis. The 4.13-Mb circular chromosome harbors biosynthetic gene clusters for the production of antimicrobial compounds. PMID:27013050

  6. Draft Genome Sequence of Streptomyces mutabilis TRM45540, Isolated from a Hypersaline Soil Sample

    OpenAIRE

    Luo, Xiaoxia; Wan, Chuanxing; Zhang, LiLi

    2015-01-01

    We report here the draft genome sequence of Streptomyces mutabilis TRM45540, a strain isolated from a soil sample from Xinjiang, China. Analysis of the genome using the bioinformatics tool antiSMASH showed the presence of many unique natural-product biosynthetic pathways.

  7. 11-Isopropylcryptolepine: A novel alkaloid isolated from cryptolepis sanguinolenta characterized using submicro NMR techniques

    Science.gov (United States)

    Hadden; Sharaf; Guido; Robins; Tackie; Phoebe; Schiff; Martin

    1999-02-01

    A new alkaloid has been isolated from extracts of the West African plant Cryptolepis sanguinolenta and identified by submicro NMR techniques as 11-isopropylcryptolepine (1). The unusual incorporation of the isopropyl group at the 11-position of the indolo[3,2-b]quinoline nucleus is suggestive of a mixed biosynthetic origin for the alkaloid. PMID:10075749

  8. 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; van der Weyden, Louise; Lund, Anders H; Wilson, Catherine H; Rance, Richard; Jonkers, Jos; van Lohuizen, Maarten; Berns, Anton; Adams, David J

    2009-01-01

    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...

  9. 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. PMID:22877740

  10. Aldehyde-Treated Porcine Skin Versus Biobrane as Biosynthetic Skin Substitutes for Excised Burn Wounds: Case Series and Review of the Literature

    OpenAIRE

    El-Khatib, H.A.; Hammouda, A.; Al-Ghol, A.; Habib, B.; Al-Basti,

    2007-01-01

    Background. The use of skin substitutes as temporary or permanent coverings has been a subject of research and study since 1500 BC. Temporary coverage of the burn wound can decrease the metabolic rate, fluid loss, pain, and colonization. The aim of this study is to review clinical experience with Biobrane and aldehyde-treated porcine skin (E.Z. Derm) as biosynthetic skin substitutes for the treatment of excised burn wounds. Methods. Fifty-two patients (42 males and 10 females) with deep derma...

  11. 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.

  12. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on.

    Science.gov (United States)

    Chomczynski, Piotr; Sacchi, Nicoletta

    2006-01-01

    Since its introduction, the 'single-step' method has become widely used for isolating total RNA from biological samples of different sources. The principle at the basis of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications. The original protocol, enabling the isolation of RNA from cells and tissues in less than 4 hours, greatly advanced the analysis of gene expression in plant and animal models as well as in pathological samples, as demonstrated by the overwhelming number of citations the paper gained over 20 years. PMID:17406285

  13. 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. PMID:26338134

  14. Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method.

    OpenAIRE

    Kovács, Katalin; Anna, Lívia; Rudnai, Péter; Schoket, Bernadette

    2011-01-01

    Bulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical...

  15. Design and methods of a social network isolation study for reducing respiratory infection transmission: The eX-FLU cluster randomized trial

    OpenAIRE

    Aiello, Allison E.; Simanek, Amanda M.; Marisa C Eisenberg; Alison R. Walsh; Brian Davis; Erik Volz; Caroline Cheng; Rainey, Jeanette J.; Amra Uzicanin; Hongjiang Gao; Nathaniel Osgood; Dylan Knowles; Kevin Stanley; Kara Tarter; Monto, Arnold S.

    2016-01-01

    Background: 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. Methodology/principal findings: 590 students in six residence halls enrolled in the eX-FLU study during a ...

  16. 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.; Gerner-Smidt, P.

    2000-01-01

    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...... S. wadsworthensis in enteric disease is needed. We conclude that a range of campylobacteria may cause infections in Denmark....

  17. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum

    Science.gov (United States)

    Huang, Wenjun; Khaldun, A. B. M.; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    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 (BCs) 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 Epimedium 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 BCs in E. sagittatum. Thus

  18. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum.

    Science.gov (United States)

    Huang, Wenjun; Khaldun, A B M; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    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 (BCs) 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 Epimedium 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 BCs in E. sagittatum. Thus

  19. Studying of Biosynthetic Pathways of 2H-labeled Purine Ribonucleoside Inosine in a Chemoheterotrophic Bacterium Bacillus subtilis B-3157 by FAB Mass-Spectrometry

    Directory of Open Access Journals (Sweden)

    Oleg Mosin

    2015-09-01

    Full Text Available This paper deals with studying biosynthetic pathways of 2H-labeled purine ribonucleoside inosine excreted into liquid microbial culture (LC by Gram-positive chemoheterotrophic bacterium Bacillus subtilis B-3157 while growing of this bacterium on heavy water (HW medium with 2% (v/v hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum B-5662 as a source of 2H-labeled growth substrates. Isolation of 2H-labeled inosine from LC was performed by adsorption/desorption on activated carbon with following extraction by 0,3 M ammonium–formate buffer (pH = 8,9, crystallization in 80% (v/v EtOH, and ion exchange chromatography (IEC on a column with AG50WX 4 cation exchange resin equilibrated with 0,3 M ammonium–formate buffer and 0,045 M NH4Cl. The investigation of deuterium incorporation into the inosine molecule by FAB method demonstrated incorporation of 5 deuterium atoms into the molecule (the total level of deuterium enrichment – 65,5 atom% 2H with 3 deuterium atoms being included into the ribose and 2 deuterium atoms – into the hypoxanthine residue of the molecule. Three non-exchangeable deuterium atoms were incorporated into the ribose residue owing to the preservation in this bacterium the minor pathways of de novo glucose biosynthesis in 2H2O-medium. These non-exchangeable deuterium atoms in the ribose residue were originated from HMP shunt reactions, while two other deuterium atoms at C2, C8-positions in the hypoxanthine residue were synthesized from [2H]amino acids, primarily glutamine and glycine, that originated from deuterated hydrolysate. A glycoside proton at -N9-glycosidic bond could be replaced with deuterium via the reaction of СО2 elimination at the stage of ribulose-5-monophosphate formation from 3-keto-6-phosphogluconic acid with subsequent proton (deuteron attachment at the С1-position of ribulose-5-monophosphate. Two other protons at C2(C3 and C4 positions in ribose residue could be

  20. FUNCTIONAL OUTCOME FOLLOWING RECONSTRUCTION FOR CHRONIC ISOLATED DORSAL DISTAL RADIOULNAR JOINT INSTABILITY BY FULKERSON-WATSON METHOD-A PROSPECTIVE STUDY

    Directory of Open Access Journals (Sweden)

    Santhamoorthy

    2014-10-01

    Full Text Available BACKGROUND: Chronic isolated distal radioulnar joint instability is a relatively rare entity. Several methods of reconstruction were available to stabilize the joint and each method has some advantage over others. We proposed to assess the functional outcome following reconstruction of chronic dorsal distal radio ulnar instability using extra articular reconstruction by Fulkerson – Watson method. AIM: To assess the functional outcome following reconstruction for chronic isolated dorsal distal radio ulnar instability using Fulkerson –Watson method. METHODS: We conducted a prospective study in five patients over three years from 2010 to 2013 with chronic isolated dorsal distal radio ulnar instability who were treated by Fulkerson-Watson method of reconstruction. All patients underwent MRI evaluation before surgery to assess ligament pathology and for adequacy of sigmoid notch. Arthroscopy performed in all patients. Functional outcomes were assessed using VAS score, quick-DASH score and Mayo wrist score at every 6 months follow-up. Radiological assessment done using plain x-rays at each follow up. RESULTS: Three patients required Arthroscopic debridement for TFCC. All five patients had achieved stability at distal radio ulnar joint after surgery and remained so till their last follow up. One patient had persistent pain near ulnar styloid. The average loss of motion for pronation was 10 degrees and supination was 3 degrees in reference to the normal side. All except one patient achieved ulnar grip strength of >90 % compared to normal side. The mean pre and postoperative VAS score, quick-DASH score, Mayo wrist score were 76.6 and 17.2, 37.3 and 11.3, 45 and 77 respectively. CONCLUSION: Though extra articular reconstruction for DRUJ by Fulkerson-Watson method is non-anatomical, the procedure is simple than intra articular reconstruction and gives similar functional outcome like intra articular reconstructions as shown by our results.

  1. A rapid and efficient method for isolating high quality DNA from leaves of carnivorous plants from the Drosera genus.

    Science.gov (United States)

    Biteau, Flore; Nisse, Estelle; Hehn, Alain; Miguel, Sissi; Hannewald, Paul; Bourgaud, Frédéric

    2012-07-01

    Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A₂₆₀/A₂₈₀ ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h. PMID:22002226

  2. Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates

    DEFF Research Database (Denmark)

    Nagy, Elisabeth; Justesen, Ulrik Stenz; Eitel, Zsuzsa;

    2015-01-01

    clearly separated the resistant and the susceptible population of B. fragilis group strains. In the case of cefoxitin only resistant population could be separated with an inhibition zone <17 mm, intermediate and susceptible isolates overlap. In conclusion, we suggest that disk diffusion can be an option...... of Bacteroides spp by comparing zone diameter results with MICs obtained earlier during an Europe-wide antibiotic susceptibility surveillance, and to propose zone diameter breakpoints, which correlate for the EUCAST MIC breakpoints. We tested 381 clinical isolates of the B. fragilis group to...... with haemin and vitamin K1. Plates were incubated at 37 °C in an anaerobic atmosphere for 24 hours. The zone diameters were read at 100% inhibition. In case of discrepant results MICs were determined by gradient test and compared with the inhibition zones on the same plate. We found a good agreement...

  3. Isolation by distance in a population of a small land snail Trochoidea geyeri: evidence from direct and indirect methods

    OpenAIRE

    Pfenninger, Markus; Bahl, Andreas; Streit, Bruno

    2006-01-01

    Population structure was estimated in a continuous population of a small land snail (Trochoidea geyeri). Mark-recapture experiments and randomly amplified polymorphic DNA analyses indicate that the population structure can be described by the isolation by distance model of Wright (1946). Estimates of density and dispersal suggest a neighbourhood size of 70-208 individuals on an area of 13-21 m 2 . A principal component analysis of the randomly amplified polymorphic DNA data reveals clinal var...

  4. Gibberellin biosynthetic inhibitors make human malaria parasite Plasmodium falciparum cells swell and rupture to death.

    Directory of Open Access Journals (Sweden)

    Tomoko Toyama

    Full Text Available Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites.

  5. Reassembled biosynthetic pathway for large-scale carbohydrate synthesis: alpha-Gal epitope producing "superbug".

    Science.gov (United States)

    Chen, Xi; Liu, Ziye; Zhang, Jianbo; Zhang, Wei; Kowal, Przemyslaw; Wang, Peng George

    2002-01-01

    A metabolic pathway engineered Escherichia coli strain (superbug) containing one plasmid harboring an artificial gene cluster encoding all the five enzymes in the biosynthetic pathway of Galalpha l,3Lac through galactose metabolism has been developed. The plasmid contains a lambda promoter, a c1857 repressor gene, an ampicillin resistance gene, and a T7 terminator. Each gene was preceded by a Shine - Dalgarno sequence for ribosome binding. In a reaction catalyzed by the recombinant E. coli strain, Galalpha 1,3Lac trisaccharide accumulated at concentrations of 14.2 mM (7.2 gL(-1)) in a reaction mixture containing galactose, glucose, lactose, and a catalytic amount of uridine 5'-diphosphoglucose. This work demonstrates that large-scale synthesis of complex oligosaccharides can be achieved economically and efficiently through a single, biosynthetic pathway engineered microorganism. PMID:17590953

  6. Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification of low temperature-induced gene overexpression

    Institute of Scientific and Technical Information of China (English)

    ZENG QingPing; ZHAO Chang; YIN LuLu; YANG RuiYi; ZENG XiaoMei; HUANG Ying; FENG LiLing; YANG XueQin

    2008-01-01

    To isolate and verify novel genes from qinghao (Artemisia annua) based on the development-specific and environment-induced transcriptomics, leaves have been harvested from the flowering A. annua plants and exposed to low temperature for isolation of total RNAs and cloning of full-length cDNAs and cDNA fragments, or expressed sequence tags (ESTs). After being sequenced and browsed for homology, these sequences have been submitted to GenBank. Among the accessed 75 sequences, 4 full-length cDNAs are highly homologous to the known A. annua genes, but 71 ESTs are absent in the sequence records of A. annua genes, in which 34 sequences are homologous to other plant genes,including 24 identified protein-coding sequences and 10 unidentified protein-coding sequences, while other 37 sequences are not present in the sequence records of any plant genes, representing the first cloned plant genes. In order to investigate the responsive patterns of A. annua genes to extreme environmental stresses, especially low temperature, the expression levels of 3 critical qinhaosu (artemisinin) biosynthetic genes, ADS, CYP71AV1 and CPR, have been measured in pre- and post-chilling A.annua seedlings cultured in vitro by semi-quantitative PCR (SQ-PCR). Consequently, ADS and CYP71AV1 genes are strongly induced by chilling, but CPR gene is not significantly affected by such treatment. Furthermore, induction of these genes by chilling can be potently suppressed by Ca2+channel inhibitor LaCl3 or Ca2+ chelator EGTA, suggesting a putative involvement of Ca2+-CaM signal transduction pathway in chilling-induced overexpression of ADS and CYP71AV1 genes. The real-time fluorescent quantitative PCR (RFQ-PCR) assay of A. annua seedlings exposed to chilling has shown that the expression level of CaM gene is up-regulated for more than 2.5 folds, thereby confirming our above inference on the relevance of Ca2+-CaM-mediated signal transduction to chilling-induced gene overexpression. Finally, 7 newly isolated A

  7. Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification of low temperature-induced gene overexpression

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To isolate and verify novel genes from qinghao (Artemisia annua) based on the development-specific and environment-induced transcriptomics, leaves have been harvested from the flowering A. annua plants and exposed to low temperature for isolation of total RNAs and cloning of full-length cDNAs and cDNA fragments, or expressed sequence tags (ESTs). After being sequenced and browsed for homol- ogy, these sequences have been submitted to GenBank. Among the accessed 75 sequences, 4 full-length cDNAs are highly homologous to the known A. annua genes, but 71 ESTs are absent in the sequence records of A. annua genes, in which 34 sequences are homologous to other plant genes, including 24 identified protein-coding sequences and 10 unidentified protein-coding sequences, while other 37 sequences are not present in the sequence records of any plant genes, representing the first cloned plant genes. In order to investigate the responsive patterns of A. annua genes to extreme envi- ronmental stresses, especially low temperature, the expression levels of 3 critical qinhaosu (artemisi- nin) biosynthetic genes, ADS, CYP71AV1 and CPR, have been measured in pre- and post-chilling A. annua seedlings cultured in vitro by semi-quantitative PCR (SQ-PCR). Consequently, ADS and CYP71AV1 genes are strongly induced by chilling, but CPR gene is not significantly affected by such treatment. Furthermore, induction of these genes by chilling can be potently suppressed by Ca2+ channel inhibitor LaCl3 or Ca2+ chelator EGTA, suggesting a putative involvement of Ca2+-CaM signal transduction pathway in chilling-induced overexpression of ADS and CYP71AV1 genes. The real-time fluorescent quantitative PCR (RFQ-PCR) assay of A. annua seedlings exposed to chilling has shown that the expression level of CaM gene is up-regulated for more than 2.5 folds, thereby confirming our above inference on the relevance of Ca2+-CaM-mediated signal transduction to chilling-induced gene overexpression. Finally, 7 newly

  8. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis

    OpenAIRE

    Liu, Chong; Colon, Brendan Cruz; Ziesack, Marika; Silver, Pamela A.; Nocera, Daniel

    2016-01-01

    Artificial photosynthetic systems can store solar energy and chemically reduce CO2. We developed a hybrid water splitting–biosynthetic system based on a biocompatible Earth-abundant inorganic catalyst system to split water into molecular hydrogen and oxygen (H2 and O2) at low driving voltages. When grown in contact with these catalysts, Ralstonia eutropha consumed the produced H2 to synthesize biomass and fuels or chemical products from low CO2 concentration in the presence of O2. This scalab...

  9. Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

    OpenAIRE

    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 ...

  10. Accessing Natural Product Biosynthetic Processes by Mass Spectrometry (Truncated Title: MS Analysis of Thiotemplate Biosynthesis)

    OpenAIRE

    Bumpus, Stefanie B.; Kelleher, Neil L.

    2008-01-01

    Two important classes of natural products are made by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). With most biosynthetic intermediates covalently tethered during biogenesis, protein mass spectrometry (MS) has proven invaluable for their interrogation. New mass spectrometric assay formats (such as selective cofactor ejection and proteomics-style LC-MS) are showcased here in the context of functional insights into new breeds of NRPS/PKS enzymes, including the firs...

  11. SELECTIVE SEPARATION OF BIOSYNTHETIC PRODUCTS BY PERTRACTION - CHALLENGE FOR THE “WHITE BIOTECHNOLOGY”

    Directory of Open Access Journals (Sweden)

    Dan Cascaval

    2010-04-01

    Full Text Available This review presents our original results on selective separation of some biosynthetic products (antibiotics, carboxylic acids, amino acids by free or facilitated pertraction (extraction and transport through liquid membranes. Selecting the optimum conditions, for all studied cases these pertraction technique simplify the technologies applied at industrial scale for separation and purification, allows to reaching high selectivity and reducing the overall cost of the products.

  12. 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.

  13. Contribution of trehalose biosynthetic pathway to drought stress tolerance of Capparis ovata Desf.

    Science.gov (United States)

    Ilhan, S; Ozdemir, F; Bor, M

    2015-03-01

    Trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Among recent findings for trehalose and its metabolism, the role of signalling in the regulation of growth and development and its potential for use as a storage energy source can be listed. The xerophytic plant Capparis ovata (caper) is well adapted to drought and high temperature stress in arid and semi-arid regions of the Mediterranean. The contribution of trehalose and the trehalose biosynthetic pathway to drought stress responses and tolerance in C. ovata are not known. We investigated the effects of PEG-mediated drought stress in caper plants and analysed physiological parameters and trehalose biosynthetic pathway components, trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), trehalase activity, trehalose and proline content in drought stress-treated and untreated plants. Our results indicated that trehalose and the trehalose biosynthetic pathway contributed to drought stress tolerance of C. ovata. Overall growth and leaf water status were not dramatically affected by drought, as both high relative growth rate and relative water content were recorded even after 14 days of drought stress. Trehalose accumulation increased in parallel to induced TPS and TPP activities and decreased trehalase activity in caper plants on day 14. Constitutive trehalose levels were 28.75 to 74.75 μg·g·FW(-1) , and drought stress significantly induced trehalose accumulation (385.25 μg·g·FW(-1) on day 14) in leaves of caper. On day 14 of drought, proline levels were lower than on day 7. Under drought stress the discrepancy between trehalose and proline accumulation trends might result from the mode of action of these osmoprotectant molecules in C. ovata. PMID:25294040

  14. Hybrid Biosynthetic Autograft Extender for Use in Posterior Lumbar Interbody Fusion: Safety and Clinical Effectiveness

    OpenAIRE

    Chedid, Mokbel K; Tundo, Kelly M; Block, Jon E; Muir, Jeffrey M

    2015-01-01

    Autologous iliac crest bone graft is the preferred option for spinal fusion, but the morbidity associated with bone harvest and the need for graft augmentation in more demanding cases necessitates combining local bone with bone substitutes. The purpose of this study was to document the clinical effectiveness and safety of a novel hybrid biosynthetic scaffold material consisting of poly(D,L-lactide-co-glycolide) (PLGA, 75:25) combined by lyophilization with unmodified high molecular weight hya...

  15. Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes

    OpenAIRE

    Bradshaw, Rosie E.; Bhatnagar, Deepak; Ganley, Rebecca J.; Gillman, Carmel J.; Brendon J. Monahan; Seconi, Janet M.

    2002-01-01

    Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen Dothistroma pini. D. pini produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic D. pini clone was identified that contained four dot genes with similarity to genes in aflatox...

  16. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes.

    Science.gov (United States)

    Li, Chun Yao; Leopold, Alex L; Sander, Guy W; Shanks, Jacqueline V; Zhao, Le; Gibson, Susan I

    2015-01-01

    Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Toward this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a "fine-tune" regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression. PMID:26483828

  17. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes

    Directory of Open Access Journals (Sweden)

    Chun Yao eLi

    2015-10-01

    Full Text Available Terpenoid indole alkaloid (TIA biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Towards this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a fine-tune regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression.

  18. Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

    OpenAIRE

    Elfahmi,

    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 Indonesian traditional herbal medicine, practised for many centuries in the Indonesian community to maintain good health and to treat diseases. The manufacturing of jamu is shifting more and more ...

  19. Artificial Chromosomes to Explore and to Exploit Biosynthetic Capabilities of Actinomycetes

    OpenAIRE

    Rosa Alduina; Giuseppe Gallo

    2012-01-01

    Actinomycetes are an important source of biologically active compounds, like antibiotics, antitumor agents, and immunosuppressors. Genome sequencing is revealing that this class of microorganisms has larger genomes relative to other bacteria and uses a considerable fraction of its coding capacity (5–10%) for the production of mostly cryptic secondary metabolites. To access actinomycetes biosynthetic capabilities or to improve the pharmacokinetic properties and production yields of these chemi...

  20. Bioactivity-guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica

    OpenAIRE

    Kersten, Roland D.; Lane, Amy L.; Nett, Markus; Richter, Taylor K. S.; Duggan, Brendan M.; Dorrestein, Pieter C.; Moore, Bradley S.

    2013-01-01

    The use of genome sequences has become routine in guiding the discovery and identification of microbial natural products and their biosynthetic pathways. In silico prediction of molecular features, such as metabolic building blocks, physico-chemical properties or biological functions, from orphan gene clusters has opened up the characterization of many new chemo- and genotypes in genome mining approaches. Here, we guided our genome mining of two predicted enediyne pathways in Salinispora trop...