Sample records for biosynthetic methods isolation

  1. Alkaloids from Pandanus amaryllifolius: Isolation and Their Plausible Biosynthetic Formation. (United States)

    Tsai, Yu-Chi; Yu, Meng-Lun; El-Shazly, Mohamed; Beerhues, Ludger; Cheng, Yuan-Bin; Chen, Lei-Chin; Hwang, Tsong-Long; Chen, Hui-Fen; Chung, Yu-Ming; Hou, Ming-Feng; Wu, Yang-Chang; Chang, Fang-Rong


    Pandanus amaryllifolius Roxb. (Pandanaceae) is used as a flavor and in folk medicine in Southeast Asia. The ethanolic crude extract of the aerial parts of P. amaryllifolius exhibited antioxidant, antibiofilm, and anti-inflammatory activities in previous studies. In the current investigation, the purification of the ethanolic extract yielded nine new compounds, including N-acetylnorpandamarilactonines A (1) and B (2); pandalizines A (3) and B (4); pandanmenyamine (5); pandamarilactones 2 (6) and 3 (7), and 5(E)-pandamarilactonine-32 (8); and pandalactonine (9). The isolated alkaloids, with either a γ-alkylidene-α,β-unsaturated-γ-lactone or γ-alkylidene-α,β-unsaturated-γ-lactam system, can be classified into five skeletons including norpandamarilactonine, indolizinone, pandanamine, pandamarilactone, and pandamarilactonine. A plausible biosynthetic route toward 1-5, 7, and 9 is proposed.

  2. 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

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    Nistri Silvia


    Full Text Available 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.

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

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    Zuiter Afnan


    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.

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

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    Hoeprich, Paul D.; Whalen, Maureen


    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.

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

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    Yuwei Sun


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

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

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    Tomova Iva


    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.

  7. 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


    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.

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

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    L. Gavrilovic


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

  9. Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway. (United States)

    Voynova, Natalya E; Rios, Sandra E; Miziorko, Henry M


    It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K(m) values for R,S-mevalonate and ATP were 41 and 339 micro M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K(i) values were 46 and 45 micro M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K(i) difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

  10. Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins (United States)

    Susca, Antonia; Proctor, Robert H.; Morelli, Massimiliano; Haidukowski, Miriam; Gallo, Antonia; Logrieco, Antonio F.; Moretti, Antonio


    The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin. PMID:27667988

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

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


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

  12. Schizosaccharomyces isolation method

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    Benito Santiago


    Full Text Available This study discusses the optimization of a selective and differential medium which would facilitate the isolation of Schizosaccharomyces (a genus with a low incidence compared to other microorganisms to select individuals from this genus for industrial purposes, especially in light of the recent recommendation of the use of yeasts from this genus in the wine industry by the International Organisation of Vine and Wine, or to detect the presence of such yeasts, for those many authors who consider them food spoilers. To this end, we studied various selective differential agents based on the main physiological characteristics of these species, such as their high resistances to high concentrations of sugar, sulfur dioxide, sorbic acid, benzoic acid, acetic acid or malo ethanolic fermentation. This selective medium is based on the genus resistance to the antibiotic actidione and its high resistance to inhibitory agents such as benzoic acid. Malic acid was used as a differential factor due to the ability of this genus to metabolise it to ethanol, which allows detecting of the degradation of this compound. Lastly, the medium was successfully used to isolate strains of Schizosaccharomyces pombe from honey and honeycombs.

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

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    Plevin, Michael J., E-mail: [CEA, Institut de Biologie Structurale Jean-Pierre Ebel (France); Hamelin, Olivier [CNRS, Laboratoire de Chimie et Biologie des Metaux (France); Boisbouvier, Jerome; Gans, Pierre [CEA, Institut de Biologie Structurale Jean-Pierre Ebel (France)


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

  14. Multilocus sequence analysis of Streptomyces griseus isolates delineating intraspecific diversity in terms of both taxonomy and biosynthetic potential. (United States)

    Rong, Xiaoying; Liu, Ning; Ruan, Jisheng; Huang, Ying


    Systematics can provide a fundamental framework for understanding the relationships and diversification of organisms. Multilocus sequence analysis (MLSA) has shown great promise for an elaborate taxonomic grouping of streptomycete diversity. To evaluate the practical significance of MLSA as a valuable systematic tool for streptomycetes, we examined six endophytic Streptomyces griseus isolates and two S. griseus reference strains possessing obvious antagonistic activities and identical 16S rRNA gene sequences, using both housekeeping genes and secondary metabolic genes. All the eight strains contained PKS-I and NRPS genes, but not PKS-II genes, and showed similar diversity in both the MLSA phylogeny based on five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) and fingerprinting of KS-AT genes. We also inferred a phylogeny based on concatenated amino acid sequences of representative KS-AT genes from the strains, which displayed a topology correlated well with those of housekeeping-gene MLSA and KS-AT fingerprinting. The good congruence observed between phylogenies based on the different datasets verified that the MLSA scheme provided robust resolution at intraspecific level and could predict the overall diversity of secondary metabolic potential within a Streptomyces species, despite somewhat of a discrepancy with antimicrobial data. It is therefore feasible to apply MLSA to dissecting natural diversity of streptomycetes for a better understanding of their evolution and ecology, as well as for facilitating their bioprospecting.

  15. Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

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    Rashel V Grindberg

    Full Text Available Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

  16. Reconstructing fungal natural product biosynthetic pathways. (United States)

    Lazarus, C M; Williams, K; Bailey, A M


    Large scale fungal genome sequencing has revealed a multitude of potential natural product biosynthetic pathways that remain uncharted. Here we describe some of the methods that have been used to explore them via heterologous gene expression. We focus on filamentous fungal hosts and discuss the technological challenges and successes behind the reconstruction of fungal natural product pathways. Optimised, efficient heterologous expression of reconstructed biosynthetic pathways promises progress in the discovery of novel compounds that could be utilised by the pharmaceutical and agrochemical industries.

  17. Biosynthetic inorganic chemistry. (United States)

    Lu, Yi


    Inorganic chemistry and biology can benefit greatly from each other. Although synthetic and physical inorganic chemistry have been greatly successful in clarifying the role of metal ions in biological systems, the time may now be right to utilize biological systems to advance coordination chemistry. One such example is the use of small, stable, easy-to-make, and well-characterized proteins as ligands to synthesize novel inorganic compounds. This biosynthetic inorganic chemistry is possible thanks to a number of developments in biology. This review summarizes the progress in the synthesis of close models of complex metalloproteins, followed by a description of recent advances in using the approach for making novel compounds that are unprecedented in either inorganic chemistry or biology. The focus is mainly on synthetic "tricks" learned from biology, as well as novel structures and insights obtained. The advantages and disadvantages of this biosynthetic approach are discussed.

  18. Tracer methods for investigating biosynthetic pathways and the metabolism of bioactive substances in plants. [Herbicides; Plant growth regulators

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    Schuette, H.R. (Akademie der Wissenschaften der DDR, Halle/Saale. Inst. fuer Biochemie der Pflanzen)


    Proceeding from the general terms of investigating the courses of reactions in plants by means of tracer methods, problems and possibilities of the methods are discussed on the basis of examples referring in particular to double labelling techniques and to the determination of the distribution of radioactivity in the resulting products. Examples of herbicides and plant growth regulators are used for describing metabolism studies.

  19. 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. (United States)

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


    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.

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

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

  1. 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 (United States)

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


    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…

  2. Characterization of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi-isozizaene albaflavenone biosynthesis and isolation of a new oxidized epi-isozizaene metabolite. (United States)

    Takamatsu, Satoshi; Lin, Xin; Nara, Ayako; Komatsu, Mamoru; Cane, David E; Ikeda, Haruo


    The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbours an orthologue (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in Streptomyces coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. Streptomyces avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in Escherichia coli catalysed the Mg²+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermitilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16 and the resulting transformants accumulated epi-isozizaene. The previously characterized oxidized epi-isozizaene metabolites (4R)- and (4S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was coexpressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-2-epi-zizaan-6β-ol which is most likely formed by oxidation of (4S)-albaflavenol.

  3. Comparison Among Three Screening Methods for Aflatoxigenlic Isolates

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    Seventy-six isolates of Aspergillus flavus,which were isolated from one hundred food stufs in Zhejiang Province,were detected with RMC,AMCS and APA methods for aflatoxigenicity.The rates of toxigenic isolates detected by the three methods were 11.8%,18.4% and 9.2% respectively.There were twenty toxigenic isolates detected by the three methods(26.3%)AMCS method was better than other methods.False negatives appeared in all methods.False positive appeared in both AMCS method and APA method.It was suggested that AMCS method may be a safe,simple and reliable method for screening toxigenic isolated if some aspects were improved.

  4. A modified method for isolation of rhein from senna. (United States)

    Mehta, Namita; Laddha, K S


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

  5. A modified method for isolation of Rhein from Senna

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    Mehta Namita


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

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

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    Liu, Q.; Manzano, D.; Tanic, N.; Pesic, M.; Bankovic, J.; Pateraki, I.; Ricard, L.; Ferrer, A.; Vos, de R.C.H.; Krol, van der A.R.; Bouwmeester, H.J.


    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 that a

  7. Emergent biosynthetic capacity in simple microbial communities.

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    Hsuan-Chao Chiu


    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

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

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


    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.

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

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    Belshoff Alex C


    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

  10. Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

    Directory of Open Access Journals (Sweden)

    Shinji Kishimoto


    Full Text Available Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid, saframycin (tetrahydroisoquinoline alkaloid, strictosidine (monoterpene indole alkaloid, ergotamine (ergot alkaloid and opiates (benzylisoquinoline and morphinan alkaloid. This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

  11. Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter. (United States)

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


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

  12. A microRNA isolation method from clinical samples

    Directory of Open Access Journals (Sweden)

    Sepideh Zununi Vahed


    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.

  13. DHAD variants and methods of screening

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, Kristen J.; Ye, Rick W.


    Methods of screening for dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed, along with DHAD variants identified by these methods. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.

  14. A Quick Method for Metarhizium anisopliae Isolation from Cultural Soils

    Directory of Open Access Journals (Sweden)

    M. A. Tajick Ghanbary


    Full Text Available Problem statement: Fungi are one of the most active members in biological community of cultural soils. Many saprophyte and facultative parasitic fungi live in soil. Metarhizium anisopliae, one of the most famous soil inhabitant entomopathogens has a virulence potential on plant and animal pests. Approach: Introducing a new method for its isolation from soil was an applied method to find it without any limitation. Metarhizium anisopliae shifts to saprophytic phase and remain alive within soil in absence of susceptible host. As a shortcut, we can transfer the fungus from soil to lab by culturing soil suspension. One hundred cultural soil samples from different regions of Iran were tested to finding Metarhizium isolates. Culturing 1:5000-1:10000 soil suspension on artificial medium containing necessary macro and micronutrients for fungal growth were resulted in isolation. Metarhizium anisopliae isolates were harvested seven days after culturing the suspensions. All isolates were inoculated in 50 mL PDB in destruxin production assay and 7 days later broth medium was filtrated by using filter paper. Culture filtrates were extracted and in bioassays they were sprayed on larva of citrus leaf miner. Results: Nine isolates of Metarhizium anisopliae were harvested. Microscopic studies showed that morphological features had complete coincidence with valid descriptions of the fungus. Bioassay confirmed that all harvested isolates secrete active and effective destruxin in broth. Conclusion: Isolation of Metarhizium by culturing the soil suspension, a useful method for more studies of the entomopathogen at different geographical regions. Native populations of this fungus had special importance in local biological control programs. This procedure was a costs- and time-effective method for pathogen isolation.

  15. Towards a Biosynthetic UAV (United States)

    Block, Eli; Byemerwa, Jovita; Dispenza, Ross; Doughty, Benjamin; Gillyard, KaNesha; Godbole, Poorwa; Gonzales-Wright, Jeanette; Hull, Ian; Kannappan, Jotthe; Levine, Alexander; Nelakanti, Raman; Ruffner, Lydia; Shumate, Alaina; Sorayya, Aryo; Ugwu, Kyla


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

  16. 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


    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

  17. 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


    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.

  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


    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. [An efficient method for isolation of mitochondrial DNA in wheat]. (United States)

    Li, Wen-Qiang; Zhang, Gai-Sheng; Wang, Kui; Niu, Na; Pan, Dong-Liang


    An efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, Dnase I treatment, lysis with SDS and proteinase K, removing protein by TE-saturated phenol/chloroform extraction and a final RNase A treatment for obtaining mtDNA. The mtDNA samples were tested using spectrophotometry and agarose gel electrophoresis. It was proved that the mtDNA isolated by this method not only have the high yield but also structural complete, and contains no impurities, such as nuclear DNA, RNA and protein. The result showed that this high quality mtDNA can be successfully used in PCR and other genetic studies. In addition, it was found that adjusting the lysis temperature has a noticeable effect on the mtDNA yield.

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

    Directory of Open Access Journals (Sweden)

    Mie Bech Lukassen


    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.

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

    DEFF Research Database (Denmark)

    Liu, Qing; Manzano, David; Tanić, Nikola


    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...... that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics. When parthenolide biosynthesis was reconstituted by transient co-expression of all pathway genes in Nicotiana benthamiana, up to 1.......4μgg-1 parthenolide was produced, mostly present as cysteine and glutathione conjugates. These relatively polar conjugates were highly active against colon cancer cells, with only slightly lower activity than free parthenolide. In addition to these biosynthetic genes, another gene encoding...

  2. Preservation methods for isolates of ascochyta blight fungi

    Directory of Open Access Journals (Sweden)

    Joanna Marcinkowska


    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.

  3. Development and Test of Methods for Fault Detection and Isolation

    DEFF Research Database (Denmark)

    Jørgensen, R.B.

    the thesis. The IPC offers prospects of repeated fault scenarios, and support studies in robustness issues. The thesis contributes with a numerical fault analysis representation, practical applications of existing methods for FDI, and a method for robust FDI for practical applications....... they are especiallu crucial for the entire operaiton of a closed loop system. The purpose of the thesis is to investigate, deveop, and verify methods for fault detection and isolation on control loop systems. An Industrial Position Controller, (IPC), laboratory setup is used as an application example throughout...

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

    Directory of Open Access Journals (Sweden)

    Gomes Luiz Humberto


    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.

  5. Degradation behaviors of nonylphenol ethoxylates by isolated bacteria using improved isolation method

    Institute of Scientific and Technical Information of China (English)

    GU Xin; ZHANG Yu; ZHANG Jing; YANG Min; Hideyuki Tamaki; Yoichi Kamagata


    Nonylphenol ethoxylate (NPEO)-degrading bacteria were isolated from activated sludge using an improved isolation method, and the corresponding degradation behaviours were investigated. Eight NPEO-degrading strains distributed in genera Pseudomonas, Sphingomonas, Sphingobium, Cupriavidus, Ralstonia, Achromobacter, and Staphylococcus were acquired. The latter five genera have never been reported for the degradation of NPEOs. Four degradation patterns were observed for the eight pure strains. In pattern A, NPEOs were converted to short-chain NPEOs and carboxylated products, while in pattern B, lower ethoxylated oligomers appeared. Nonylphenol monoethoxylate was the main product in pattern C, while in pattern D ethoxylated units was oxidized but not shortened. Pattern C and D have not yet been reported.

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

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


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

  7. Advances in Isolation Methods for Spermatogonial Stem Cells. (United States)

    Zhang, Rui; Sun, Jin; Zou, Kang


    Stem cell research has led to many remarkable achievements in recent years, but progress in the study of spermatogonial stem cells (SSCs) has been relatively slow, partly due to the slow development of techniques for spermatogonial stem cell isolation. The major accomplishments of SSC sorting and identification occurred approximately 10 years ago, and since that time, these techniques have been widely used without major improvements. In this article, we briefly introduce the biological properties of SSCs before reviewing the development of sorting techniques for SSCs in the past decades. We then summarize recent achievements in SSC sorting and finally discuss the advantages and disadvantages of SSC isolation methods, to provide new insight into techniques and research related to spermatogonial stem cells and promote the development of reproductive biology.

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

    DEFF Research Database (Denmark)

    Liu, Chengwei; Tagami, Koichi; Minami, Atsushi;


    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....

  9. An Effective Method for Total RNA Isolation from Bamboo

    Institute of Scientific and Technical Information of China (English)

    GAO Zhimin; LI Xueping; LI Lubin; PENG Zhenhua


    Trizol reagent was used for RNA isolation from fresh leaves of Dendrocalamopsis oldhami, Bambusa ventricosa and Phyllostachys aureosulcata cv. Pekinensis. The extracted RNA from leaves had the normal ultraviolet absorption, the value of OD26(/OD280 varied between 1.8-2.0. The 28s rRNA was more than two times brighter than 18s rRNA in electrophoresis. These results indicated that the total RNA was complete and not degraded. According to our experiments, RNA could be obtained simply with this method, and can be used for molecular manipulation such as cDNA synthesis and gene cloning.


    Energy Technology Data Exchange (ETDEWEB)

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


    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

  11. A method for isolation of milk fat globules. (United States)

    Patton, S; Huston, G E


    The traditional procedure for isolating milk fat globules involves repeated cycles of centrifuging to obtain globules and redispersion of them in fresh buffer to eliminate other milk components. We have evaluated a simpler, less manipulative method whereby globules are centrifuged out of the milk and through an overlying buffer layer. Human milk samples ranging from 0.1 to 35 ml were centrifuged at 1500 X g for 20 min after deposition under a suitable quantity of buffer. This yielded purified globules, in less time, which could be dispersed more satisfactorily than those by the traditional procedure. Protein, phospholipid and cholesterol contents of globules by the two methods were quite similar. A lower protein content (10.4 vs 13.2 mg/g of lipid) was characteristic of globules prepared by the multiple wash method. However, large differences could not be seen in gel electrophoresis patterns of the proteins. By using plastic centrifuge tubes, tube freezing and cleavage just below the globule layer enables clean separation of globule and nonglobule phases for analysis of milk component distributions. Macro (5 to 35 ml of sample) and micro (200 microliter or less) versions of the method are described. Limited trials showed that the method can be applied satisfactorily to cow's and goat's milks, but for highly pure globules a deeper buffer column than that used with human milk is required because of their much higher casein content.

  12. Characterization of the fumonisin B2 biosynthetic gene cluster in Aspergillus niger and A. awamori. (United States)

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

  13. Simple method of isolating humic acids from organic soils (United States)

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


    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

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

    DEFF Research Database (Denmark)

    Gelso, Esteban R.; Blanke, Mogens


    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 paper develops algorithms for investigation of the possibilities of active structural isolation and it offers illustrative examples and a larger case study to explore the properties of active structural isolability ideas....

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

    CERN Document Server

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


    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.

  16. Isolation and identification methods of Rothia species in oral cavities. (United States)

    Tsuzukibashi, Osamu; Uchibori, Satoshi; Kobayashi, Taira; Umezawa, Koji; Mashimo, Chiho; Nambu, Takayuki; Saito, Masanori; Hashizume-Takizawa, Tomomi; Ochiai, Tomoko


    Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable

  17. Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast. (United States)

    Tang, Man-Cheng; Lin, Hsiao-Ching; Li, Dehai; Zou, Yi; Li, Jian; Xu, Wei; Cacho, Ralph A; Hillenmeyer, Maureen E; Garg, Neil K; Tang, Yi


    The structural diversity and biological activities of fungal indole diterpenes (IDTs) are generated in large part by the IDT cyclases (IDTCs). Identifying different IDTCs from IDT biosynthetic pathways is therefore important toward understanding how these enzymes introduce chemical diversity from a common linear precursor. However, IDTCs involved in the cyclization of the well-known aflavinine subgroup of IDTs have not been discovered. Here, using Saccharomyces cerevisiae as a heterologous host and a phylogenetically guided enzyme mining approach, we combinatorially assembled IDT biosynthetic pathways using IDTCs homologues identified from different fungal hosts. We identified the genetically standalone IDTCs involved in the cyclization of aflavinine and anominine and produced new IDTs not previously isolated. The cyclization mechanisms of the new IDTCs were proposed based on the yeast reconstitution results. Our studies demonstrate heterologous pathway assembly is a useful tool in the reconstitution of unclustered biosynthetic pathways.

  18. miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods. (United States)

    Nagy, Zsófia Brigitta; Wichmann, Barnabás; Kalmár, Alexandra; Barták, Barbara Kinga; Tulassay, Zsolt; Molnár, Béla


    MiRNA remain stable for detection and PCR-based amplification in FFPE tissue samples. Several miRNA extraction kits are available, however miRNA fraction, as part of total RNA can be isolated using total RNA purification methods, as well. Our primary aim was to compare four different miRNA and total RNA isolation methods from FFPE tissues. Further purposes were to evaluate quantitatively and qualitatively the yield of the isolated miRNA. MiRNAs were isolated from normal colorectal cancer FFPE specimens from the same patients. Two miRNA isolation kits (High Pure miRNA Isolation Kit, miRCURY™ RNA Isolation Kit) and two total RNA isolation kits were compared (High Pure RNA Paraffin Kit, MagNA Pure 96 Cellular RNA LV Kit). Quantity and quality were determined, expression analysis was performed by real-time PCR using qPCR Human Panel I + II (Exiqon) method detecting 742 human miRNAs in parallel. The yield of total RNA was found to be higher than miRNA purification protocols (in CRC: Ex: 0203 ± 0021 μg; HPm: 1,45 ± 0,8 μg; HPp: 21,36 ± 4,98 μg; MP: 8,6 ± 5,1 μg). MiRNAs were detected in lower relative quantity of total RNA compared to the miRNA kits. Higher number of miRNAs could be detected by the miRNA isolation kits in comparison to the total RNA isolation methods. (Ex: 497 ± 16; HPm: 542 ± 11; HPp: 332 ± 36; MP: 295 ± 74). Colon specific miRNAs (miR-21-5p;-34-5p) give satisfying results by miRNA isolation kits. Although miRNA can be detected also after total RNA isolation methods, for reliable and reproducible miRNA expression profiling the use of miRNA isolation kits are more suitable.

  19. Isolation method (direct plating or enrichment) does not affect antimicrobial susceptibility of Campylobacter from chicken carcasses (United States)

    To determine if Campylobacter isolation method influenced antimicrobial susceptibility results, the minimum inhibitory concentrations (MIC) of nine antimicrobials were compared for 291 pairs of Campylobacter isolates recovered from chicken carcass rinse samples using direct plating and an enrichment...

  20. An improved method to isolate lichen algae by gel filtration. (United States)

    Pérez, M J; Vicente, C; Legaz, M E


    Photobiont cells of the lichen Evernia prunastri have completely been separated from their fungal partner by filtration through a bed of Sepharose 2B. Both mannitol and ribitol have been quantified by gas-liquid chromatography in the different steps of the isolation procedure. Absence of mannitol, which is exclusively produced by the mycobiont, has been used as the best probe to monitor isolation.

  1. Minimum Information about a Biosynthetic Gene cluster

    NARCIS (Netherlands)

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


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

  2. Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria

    KAUST Repository

    Ross, Avena C.


    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.

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

    Directory of Open Access Journals (Sweden)

    Oberoi A


    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.

  4. Comparison of Arcobacter isolation methods, and diversity of Arcobacter spp. in Cheshire, United Kingdom. (United States)

    Merga, J Y; Leatherbarrow, A J H; Winstanley, C; Bennett, M; Hart, C A; Miller, W G; Williams, N J


    The aims of this study were, firstly, to compare five published methods for the isolation of Arcobacter spp. from animal feces in order to determine the most sensitive and specific method. Second, we analyzed the resulting isolates by multilocus sequence typing (MLST) in order to investigate the diversity of the isolates recovered. Third, we investigated the ability to recover Arcobacter spp. from frozen fecal samples. Seventy-seven fecal samples from cattle, sheep, and badgers were subjected to five isolation methods, based on published methods for the isolation of Arcobacter and Campylobacter spp. Thirty-nine Arcobacter butzleri isolates were analyzed using a multilocus sequence typing scheme. The survival of Arcobacter spp. in frozen samples was investigated by freezing the fecal samples at -80°C for 7 days and then applying the same five isolation methods. The most sensitive and specific method used an Arcobacter-specific broth in conjunction with modified charcoal cefoperazone deoxycholate agar (mCCDA) with added antibiotics. Freezing of fecal samples led to a reduction in the recovery of Arcobacter spp. by approximately 50%. The 39 allelic profiles obtained by MLST could be divided into 11 sequence types (STs). We have identified the most sensitive and specific method for the isolation of Arcobacter spp. from animal feces and demonstrated that the freezing of fecal samples prior to isolation reduces arcobacter recovery. MLST analysis of the isolates revealed a high level of diversity.

  5. Methods and Devices for Micro-Isolation, Extraction, and/or Analysis of Microscale Components (United States)

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


    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.

  6. A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types. (United States)

    Patel, Ketan D; Bhanshali, Forum C; Chaudhary, Avani V; Ingle, Sanjay S


    New or more efficient methodologies having different principles are needed, as one method could not be suitable for isolation of organisms from samples of diverse types and from various environments. In present investigation, growth kinetics study revealed a higher germination rate, a higher growth rate, and maximum sporulation of Bacillus thuringiensis (Bt) compared to other Bacillus species. Considering these facts, a simple and efficient enrichment method was devised which allowed propagation of spores and vegetative cells of Bt and thereby increased Bt cell population proportionately. The new enrichment method yielded Bt from 44 out of 58 samples. Contrarily, Bt was isolated only from 16 and 18 samples by sodium acetate selection and dry heat pretreatment methods, respectively. Moreover, the percentages of Bt colonies isolated by the enrichment method were higher comparatively. Vegetative whole cell protein profile analysis indicated isolation of diverse population of Bt from various samples. Bt strains isolated by the enrichment method represented novel serovars and possibly new cry2 gene.

  7. A comparison of Arcobacter isolation methods and the diversity of Arcobacter spp. in Cheshire, UK.__________________________________________ (United States)

    Aims: The aims of this study were firstly to compare five published methods for the isolation of Arcobacter spp. from animal faeces in order to determine the most sensitive and specific method. Secondly, we analyzed the resulting isolates by multi-locus sequence typing (MLST) in order to investiga...

  8. Optimal fractionation and bioassay plans for isolation of synergistic chemicals: The subtractive-combination method. (United States)

    Byers, J A


    Studies of chemical ecology of an organism are founded on the isolation and identification of a semiochemical, often comprised of two or more synergistic compounds (each Synergist alone has little activity, but presented together they are bioactive). Chromatographie fractionation and bioassay methods of binary splitting, additive combination, and subtractive combination are compared for efficiency in isolating synergists. Formulas are derived for the latter two methods that calculate the expected number of bioassay tests required for isolation of from two to five synergists from biological extracts with any number of compounds, depending on the number of initial (major) Chromatographic fractions. A computer program based on the formulas demonstrates the superiority of the subtractive-combination method. Simulations with the program were used to determine the optimal number of initial fractions for the additive- and subtractive-combination methods when isolating two to five synergists from extracts of from 25 to 1200 compounds. Methods of bioassay, isolation, identification, and field testing of semiochemicals are discussed.

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

    DEFF Research Database (Denmark)

    Podolska, Agnieszka; Kaczkowski, Bogumil; Litman, Thomas


    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 microRNA...... 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....

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

    Directory of Open Access Journals (Sweden)



    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.

  11. Distinct mechanisms for spiro-carbon formation reveal biosynthetic pathway crosstalk. (United States)

    Tsunematsu, Yuta; Ishikawa, Noriyasu; Wakana, Daigo; Goda, Yukihiro; Noguchi, Hiroshi; Moriya, Hisao; Hotta, Kinya; Watanabe, Kenji


    Spirotryprostatins, an indole alkaloid class of nonribosomal peptides isolated from Aspergillus fumigatus, are known for their antimitotic activity in tumor cells. Because spirotryprostatins and many other chemically complex spiro-carbon-bearing natural products exhibit useful biological activities, identifying and understanding the mechanism of spiro-carbon biosynthesis is of great interest. Here we report a detailed study of spiro-ring formation in spirotryprostatins from tryprostatins derived from the fumitremorgin biosynthetic pathway, using reactants and products prepared with engineered yeast and fungal strains. Unexpectedly, FqzB, an FAD-dependent monooxygenase from the unrelated fumiquinazoline biosynthetic pathway, catalyzed spiro-carbon formation in spirotryprostatin A via an epoxidation route. Furthermore, FtmG, a cytochrome P450 from the fumitremorgin biosynthetic pathway, was determined to catalyze the spiro-ring formation in spirotryprostatin B. Our results highlight the versatile role of oxygenating enzymes in the biosynthesis of structurally complex natural products and indicate that cross-talk of different biosynthetic pathways allows product diversification in natural product biosynthesis.

  12. Methods for isolation and viability assessment of biological organisms (United States)

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


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

  13. Comparison of Six Culture Methods for Salmonella Isolation from Poultry Fecal Samples

    Directory of Open Access Journals (Sweden)

    Morshed, R. (PhD


    Full Text Available Background and Objective: Salmonellosis is one of the most important food-borne bacterial zoonotic diseases worldwide, and poultry and its products are the major sources for salmonella transmission to human. Isolation of Salmonella enterica from poultry needs bacteriologic enrichment and selected cultures of fecal samples. In this study, different culture methods for the isolation of salmonella from fecal samples were compared. Material and Methods: Forty- five positive samples from infected farms and 45 negative samples from normal farms were processed using enrichment media including tetrathionate broth, selenite cistine and Rappaport-Vassiliadis. Then the samples were incubated in selective cultures, and after 24 h, their results were compared with standard method. Results: Specificity of all methods for salmonella isolation was 100%, and salmonella was not isolated from the negative samples. The highest susceptibility was related to the method in which the sample first in Selenite cistine and later in Rappaport-Vassiliadis was enriched (100%. Enrichment in Rappaport-Vassiliadis could isolate 41 salmonella from 45 positive samples (91% while the result of enrichment in tetrathionate was 6 isolates (13.3%. Conclusion: This study shows that enrichment in selenite cistine and then in Rappaport-Vassiliadis is currently the best method for isolating salmonella from fecal samples of poultry. Key words: Salmonella; Bacteriologic Culture; Diagnosis; Isolation; Enrichment; Poultry

  14. A simple and efficient DNA isolation method for Salvia officinalis. (United States)

    Aleksić, Jelena M; Stojanović, Danilo; Banović, Bojana; Jančić, Radiša


    We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4 M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6 μg DNA g(-1) of dry leaf tissue, and the absorbance ratios 260/280 and 260/230 nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.

  15. A simple method for RNA isolation from various tissues of the tree Neolamarckia cadamba



    Plant tissues contain abundant polysaccharides, phenolic compounds and other metabolites, which makes it difficult to isolate high-quality RNA from them. In addition, Neolamarckia cadamba contains large quantities of other components, particularly RNA-binding alkaloids, which makes the isolation even more challenging. Here, we describe a concise and efficient RNA isolation method that combines the cetyltrimethyl ammonium bromide (CTAB) and Plant RNA Kit (Omega) protocols. Gel electrophoresis ...

  16. Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae

    Directory of Open Access Journals (Sweden)

    A.L.C. Darini


    Full Text Available In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology. Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae.

  17. Noninvasive method of DNA isolation from fecal epithelial tissue of dairy animals. (United States)

    Chandra De, Bidhan; Patra, Mahesh Chandra; Kumar, Sushil; Brahma, Biswajit; Goutam, Devika; Jaiswal, Latika; Sharma, Ashutosh; De, Sachinandan


    A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.

  18. Characterization of the promoter region of biosynthetic enzyme genes involved in berberine biosynthesis in Coptis japonica

    Directory of Open Access Journals (Sweden)

    Yasuyuki Yamada


    Full Text Available The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs, a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1 were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA and by a chromatin immunoprecipitation (ChIP assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.

  19. 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


    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

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

    Directory of Open Access Journals (Sweden)

    Claudiu Iavornic


    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.

  1. Principal methods for isolation and identification of soil microbial communities. (United States)

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


    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.

  2. Disk Inoculum-Solid Medium Method To Test Carbon and Nitrogen Assimilation by Yeast Isolates


    Moore, Kerry J.; Johnson, Michael G.; McClary, Shane P.


    Carbon and nitrogen assimilation for 50 yeasts isolated from White Riesling fermentation were tested by using a disk inoculum-solid medium method. This method was quicker and gave results comparable to the conventional liquid medium methods. Yeast characteristics (growth response, pigment production, morphology) could also be compared with this method.

  3. 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.

  4. 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.


    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.

  5. The biosynthetic gene cluster for the beta-lactam carbapenem thienamycin in Streptomyces cattleya. (United States)

    Núñez, Luz Elena; Méndez, Carmen; Braña, Alfredo F; Blanco, Gloria; Salas, José A


    beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics. This involves the participation of a beta-lactam synthetase. Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya. Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

  6. A rapid and reliable method for the clonal isolation of Acanthamoeba from environmental samples

    Directory of Open Access Journals (Sweden)

    Janice Zanella


    Full Text Available Acanthamoeba are abundant in a wide range of environments, and some species are responsible for cutaneous infections, keratitis, and granulomatous amoebic encephalitis (GAE. The conventional detection and isolation of amoeba from clinical and environmental samples involves sampling and culture on non-nutrient Ágar medium. Although efficient, this system requires several transfers in order to eliminate contaminants, and is not appropriate for the isolation of individual amoeba from samples with a biodiverse community. In this study we propose an alternative method for the isolation of monocystic clones of Acanthamoeba. The propose method involves sampling, enrichment, encystment induction, and direct cysts micromanipulation and culture on Ágar plates.

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

    Directory of Open Access Journals (Sweden)

    Massimiliano Ferraioli


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

  8. Metabolic engineering of biosynthetic pathway for production of renewable biofuels. (United States)

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


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

  9. A New Method for Isolating and Purifying Natural Drug Taxol

    Institute of Scientific and Technical Information of China (English)

    肖剑; 韩金玉; 等


    A pharmacological interaction target(PFI)method for solving the difficult problem in the separation of taxol from cephalonmanin was proposed.A two-phase extraction technique was used to carry out the PIT separation process.The effects of buffer,temperature and protein on the separation were investigated.Feasible disassembly conditions were also discussed.The final purity of taxol can reach 95% or higher.

  10. A Simple Method for Isolation of Neoblasts from Planaria

    Directory of Open Access Journals (Sweden)

    Hamed Chitsazan


    Full Text Available Objective: Freshwater planarians were used as models for studying metazoan regenerationand stem cell biology. Here a simple, fast and high throughput method for extracting theirstem cells (neoblasts is represented.Materials and Methods: Specimens of the Dugesia sp with an average length of 18 mmwere homogenized by a glass Dounce tissue grinder which contained about 1 ml of planariansaline solution. The extracted suspension was serially filtered by 60, 41, 30, 20 and 11 μmnylon meshes. In order to obtain purified neoblasts in the final suspension; this suspensionhas been compared with a cell suspension from 30 Gy irradiated worms. Hoechst 33342was used to determine cells from non-cellular particles; methylene blue and propidium iodidewere used to detect the number of dead cells in each extraction.Results: About 2.6-3 million cells were extracted from 10-12 worms. Flow cytometry analysisshowed about 83% of the extracted particles were cells. In suspensions from irradiatedanimals, about 50% of the cells were absent, the final suspension contained about 62-66%neoblasts and about 17% non-cellular particles. When these extracts were treated with distilledwater to destroy the cells, only rabdites and chitinous spines of the parenchyma wereobserved in the extract.Conclusion: Results show that the purity of neoblasts in the final suspension is about 66%.Non-cellular particles have a carbohydrate nature and, therefore, this extraction method iscompletely compatible with molecular (e.g. proteomics and transcriptomics and cellularmethods (e.g. neoblast culture.

  11. PCR ITS-RFLP: A useful method for identifying filamentous fungi isolates on grapes. (United States)

    Diguta, C F; Vincent, B; Guilloux-Benatier, M; Alexandre, H; Rousseaux, S


    Restriction digestion analysis of the ITS products was tested as an easy method to identify isolates of filamentous fungi on grapes. Endonucleases SduI, HinfI, MseI, HaeIII were used. Endonucleases BfmI, Cfr9I, Hpy188I, MaeII or PspGI were used as necessary to complete discrimination. The 43 species studied generated 42 different composite profiles. Only the species P. thomii and P. glabrum gave the same composite profile. 96.3% strains tested could be differentiated to the species level with only four enzymes. Hundred ninety nine strains of filamentous fungi were isolated from various vineyards in Burgundy and identified by this method. Penicillium (58.5%) was the genus the most frequently isolated and no strains of the genus Aspergillus was isolated. P. spinolusum was the most isolated species of Penicillium (22.70%). The species C. cladiosporioides, B. cinerea, E. nigrum, A. alternata, T. koningiopsis, P. diplodiella, C. herbarum, A. alternatum, T. cucumeris and F. oxysporum were also isolated. This technique is a rapid and reliable method appropriate for routine identification of filamentous fungi. This can be used to screen large numbers of isolates from various environments in a short time. This is the first exhaustive study of fungal diversity at species level in vineyard.

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

    DEFF Research Database (Denmark)

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


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

  13. Differences in vancomycin MIC among MRSA isolates by agar dilution and E test method

    Directory of Open Access Journals (Sweden)

    K Tandel


    Full Text Available In this study, the correlation between vancomycin minimum inhibitory concentration (MIC obtained by the E test technique and the Clinical And Laboratory Standards Institute (CLSI agar dilution method was evaluated. A total of 53 Methicillin Resistant Staphylococcus aureus (MRSA strains were tested by both the methods in the present study. MICs of vancomycin obtained by the E test method were consistently higher (+0.5 to 2 log2 dilutions than those obtained by the agar dilution method. Out of 53 MRSA isolates, 49 isolates showed higher MIC results by E test than by agar dilution method. Three isolates showed same MIC result by both methods. Since many studies have demonstrated increased clinical failure with MRSA isolates for which vancomycin MICs are increased (>1 μg/ml but still within the susceptibility range (≤ 2 μg/ml, our findings suggest the requirement to re-look into the breakpoints for vancomycin for determining sensitivity of MRSA isolates. Guidelines should also specify the method to be used for determining the MIC.

  14. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies


    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin


    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen)...

  15. Comparison of three phenotypic and deoxyribonucleic acid extraction methods for isolation and Identification of Nocardia spp

    Directory of Open Access Journals (Sweden)

    Jamshid Faghri


    Conclusions: These bacteria are important in immune deficient patients such as cancer patients, transplant recipients, tuberculosis; acquired immunodeficiency syndrome etc., Their affluence is unsteady in different zones of the world. In this study, among the three phenotypic methods for the isolation of Nocardia slip-buried method was better than other methods. Among DNA extraction techniques, DNA extraction by microwave method would be selective method for DNA extraction of Nocardia spp. compared with others techniques.

  16. An extension of the Coconut Cream Agar method to screen Penicillium citrinum isolates for citrinin production. (United States)

    Mohamed, S; Flint, S; Palmer, J; Fletcher, G C; Pitt, J I


    A simple and rapid screening method was developed for the detection of citrinin in fungal cultures using Coconut Cream Agar (CCA) described previously for detecting aflatoxin and ochratoxin A. Fifteen isolates of Penicillium citrinum were inoculated onto CCA and incubated at 25 and 30°C for 10 days. All isolates produced a distinct yellow green fluorescence on CCA when the reverse side of the agar plates were viewed under long wavelength UV light. Detection was optimal at 25°C after four to 5 days of incubation. Isolates positive by the CCA method also tested positive for citrinin production by the TLC agar plug method after growth on CCA, Czapek yeast extract agar and yeast extract sucrose agar. Control cultures were negative by both methods, indicating that the CCA Petri dish method was suitable for screening cultures for citrinin production.


    Institute of Scientific and Technical Information of China (English)

    Huo Rui; Shi Yin


    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.

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

    Directory of Open Access Journals (Sweden)

    Mahmoudi Rad M


    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

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

    Directory of Open Access Journals (Sweden)

    Afreenish Hassan


    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.

  20. Influence of DNA isolation method on the investigation of archaeal diversity and abundance in biogas plants. (United States)

    Theiss, Juliane; Rother, Michael; Röske, Kerstin


    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.

  1. Epidemiological typing of clinical isolates of Achromobacter xylosoxidans: comparison of phenotypic and genotypic methods. (United States)

    Kaur, M; Ray, P; Bhatty, M; Sharma, M


    The purpose of this paper is to evaluate the utility of different typing methods for Achromobacter xylosoxidans clinical isolates. Ninety-two blood culture isolates of A. xylosoxidans subsp. xylosoxidans were collected over a 25-month period. The typeability, discriminatory power and reproducibility of commonly used phenotypic and genotypic methods, such as resistotyping, plasmid profiling, whole-cell protein fingerprinting, random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE), were compared. All 92 isolates were typeable by all of the methods used, with comparable reproducibility. PFGE showed the highest discriminatory power (98.9%), but whole-cell protein profiling showed better correlation with epidemiological data without significant loss in discriminatory power (94%). Whole-cell protein profiling is a reliable epidemiological tool for the analysis of A. xylosoxidans; PFGE is the most discriminatory.

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

    DEFF Research Database (Denmark)

    Cirera Salicio, Susanna


    high quality RNA with a reasonable yield from adipose tissue. The aim of this study was to provide an optimized protocol for isolating total RNA from adipose tissue. This was achieved by combining the advantages of the two routinely used methods, TRI Reagent(R) and miRNeasy. FINDINGS: The mi......-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.......Background: RNA extraction is a crucial step for monitoring gene expression. Poor RNA quality (including degradation and remaining impurities) can result in misleading results. Isolation of RNA from animal tissues with high lipid content can be challenging. Especially, it is not trivial to isolate...

  3. Cloning of full genome sequence of hepatitis E virus of Shanghai swine isolate using RACE method

    Directory of Open Access Journals (Sweden)

    Mou Jing


    Full Text Available Abstract Genotype 4 hepatitis E virus (HEV was reportedly transmitted freely between humans and swine in eastern China. The full-length genomic sequence of Shanghai swine isolate (SH-SW-zs1 recovered from feces sample of a pig which was infected with HEV RNA positive swine serum was determined using RT-PCR and RACE (Rapid Amplification of cDNA Ends methods. The full genome of the SH-SW-zs1 isolate was 7265 nucleotides in length and phylogenetic analysis indicated that this isolate belonged to genotype 4. Comparison of the 3' UTR sequence with the corresponding regions of other 38 HEV strains from different region revealed that the Shanghai swine isolate is 21–49 bp longer than the other stains.

  4. 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


    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.

  5. Pyrimidine biosynthetic pathway of Baccillus subtilis. (United States)

    Potvin, B W; Kelleher, R J; Gooder, H


    Biochemical and genetic data were obtained from a series of 51 Pyr- strains of Bacillus subtilis. The observed enzymatic deficiencies allowed the mutants to be placed into 12 clases, some of which represent defects in more than one of the six known pyrimidine biosynthetic enzymes. Mapping analysis by transformation has shown that all the Pyr- mutations are located in a single small area of the B. subtilis genome. A correlation of the biochemical defects and the genetic data has been made. Those mutations conferring similar enzymatic deficiencies were found to be contiguous on the B. subtilis map. Regulatory aspects of the pyrimidine pathway have also been investigated and are compared to previously reported results from other organisms. Evidence is presented which bears upon the possible physical association of the first three enzymes and the association of at least some of the enzymes of this pathway with particulate elements of the cell. A model for the organization of the enzymes is presented with dihydroorotate dehydrogenase as the central enzyme in a proposed aggregate.

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

    Directory of Open Access Journals (Sweden)

    de Folter Stefan


    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.

  7. Comparison of serological and sequence-based methods for typing feline calcivirus isolates from vaccine failures. (United States)

    Radford, A D; Dawson, S; Wharmby, C; Ryvar, R; Gaskell, R M


    Feline calicivirus (FCV) can be typed by exploiting antigenic differences between isolates or, more recently, by the sequence analysis of a hypervariable region of the virus's capsid gene. These two methods were used to characterise FCV isolates from 20 vaccine failures which occurred after the use of a commercial, live-attenuated vaccine. Using virus neutralisation, the isolates showed a spectrum of relatedness to the vaccine; depending on the criterion adopted for identity, 10 to 40 per cent of them appeared to be similar to the vaccine virus. Using sequence analysis, the isolates fell into one of two categories; 20 per cent had a similar sequence to the vaccine (0-67 to 2-67 per cent distant), and the remainder had a dissimilar sequence (21-3 to 36-0 per cent distant). Sequence analysis identified one cat that appeared to be infected with two distinct FCVs. The serological and sequence-based typing methods gave the same result in 80 to 95 per cent of individual cases, depending on the criterion adopted for serological identity. It is suggested that molecular typing is a more definitive method for characterising the relatedness of FCV isolates.

  8. 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.

  9. A combined dynamic analysis method for geometrically nonlinear vibration isolators with elastic rings (United States)

    Hu, Zhan; Zheng, Gangtie


    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.

  10. Novel Campylobacter isolation method using hydrophobic grid membrane filter and semisolid medium. (United States)

    Valdivieso-Garcia, Alfonso; Harris, Kathleen; Riche, Edward; Campbell, Stephanie; Jarvie, Anne; Popa, Maria; Deckert, Anne; Reid-Smith, Richard; Rahn, Kris


    Culture procedures for isolation of thermophilic campylobacters from food matrices are complex, labor intensive, and time-consuming. Most available methods include the use of antibiotics as selective agents to prevent the growth of competing microflora. A simple procedure for isolation of thermophilic campylobacters after enrichment in Rosef's enrichment broth was developed using a hydrophobic grid membrane filter (HGMF) on semisolid medium (SSM). SSM contains no antibiotics, and the HGMF physically separates Campylobacter from the enrichment broth, allowing isolation based on differential motility. The HGMF-SSM method was compared to the Agriculture and Agri-Food Canada Food Safety Procedures Manual (FSPM-10) method (Isolation of Thermophilic Campylobacters from Fresh Pork, Beef Veal, Poultry and Ready-to-Eat Meat Products), which includes the use of selective antibiotics. During the initial study, after enrichment the HGMF-SSM method yielded pure cultures of campylobacters after 16 to 18 h (overnight) compared with 48 h for the FSPM-10 method. Ninety-four turkey samples collected at local retail stores and 38 frozen pig fecal samples were processed by both methods. Thirty-five samples (26.5%) were positive by the HGMF-SSM method; 24 (18.2%) of these positive samples contained Campylobacter jejuni and 11 (8.3%) contained Campylobacter coli. With the FSPM-10 method, 25 samples (18.9%) were positive: 21 (15.9%) with C. jejuni and 4 (3%) with C. coli. For a subsequent field study, only the HGMF-SSM method was used to isolate Campylobacter from 1,200 chicken samples and 454 turkey samples sold at retail. Analysis of five subisolates from various samples indicated that only one type of Campylobacter was recovered by the HGMF-SSM method, as ascertained by MICs for 10 antimicrobials, sequencing of the short variable region of the flaA gene, and fingerprinting based on amplified fragment length polymorphism. The absence of antibiotics in the SSM may explain the higher

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

    Directory of Open Access Journals (Sweden)

    K Beheshti-Maal


    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.

  12. 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

  13. Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway. (United States)

    Hodges, R L; Kelkar, H S; Xuei, X; Skatrud, P L; Keller, N P; Adams, T H; Kaiser, R E; Vinci, V A; McGilvray, D


    Echinocandin B (ECB), a lipopolypeptide used as a starting material for chemical manufacture of the anti-Candida agent LY303366, is produced by fermentation using a strain of Aspergillus nidulans. In addition to ECB, the wild-type strain also produces a significant level of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Characterization of a mutant designated A42355-OC-1 (OC-1), which is blocked in ST biosynthesis, was the result of a chromosomal translocation. The chromosomal regions containing the breakpoints of the translocation were isolated and DNA sequencing and PCR analysis of the chromosomal breakpoints demonstrated the translocation occurred within the stcW gene of the ST biosynthetic pathway, resulting in disruption of the open reading frame for this gene. Biochemical feeding studies indicate the involvement of this gene product in the conversion of averufin to 1-hydroxy versicolorone. This work demonstrates an effective synergy between classical strain improvement methods and molecular genetics.

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

    Directory of Open Access Journals (Sweden)

    Baskaran, R.


    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.

  15. Expression of Bacillus Species Isolated Phytase Gene from Soil by PCR Method

    Directory of Open Access Journals (Sweden)

    Nazemi, A. (PhD


    Full Text Available Background and Objective: Recognizing and using of isolated phytase in the soil microorganisms are paramount importance to produce the Phytase enzyme utilized commercially in different industries. This study was conducted to recognize different bacillus species which are Phytase producers and detection of the gene that can produce this enzyme. Material and Methods: Soil samples were gathered through different parts of mountainous areas. The early isolation of bacillus was carried out in Bacillus Medium Agar. After isolating the bacteria and genome extraction, the responsible gene of enzyme producer recognized and amplified by PCR method. The size of this protein and the optimal production situation in supplemental exploitation such as SDS-PAGE and the enzymatic activity of its size were evaluated. Results: Of 40 samples, one bacterium secreting Phytase enzyme was isolated. This bacterium was sequenced and recognized Bacillus Sobtlis species that is classified in STR Genus. The size of protein phytase produced by this gene was about 45 KD and the enzyme activity at 55 degrees was measured about 5.65 in wavelength of 415 NM. The phytase gene with the size of 1200 bp was propagated. Conclusion: the microorganisms, in natural conditions, produce Phytase enzyme in limited amount and with the quality appropriate to microorganisms. Thus, isolating the bacilli producing Phytase enzyme and purifying this protein are highly significant. Key words: Bacillus Subtilis; Phytase; SDS-PAGE; Enzymatic Activity; Polymerization Chain Reaction

  16. Microorganism genomics, compositions and methods related thereto (United States)

    Handelsman, Jo; Goodman, Robert M.; Rondon, Michelle R.


    The present invention provides methods and compositions for accessing, in a generally unbaised manner, a diverse genetic pool for genes involved in biosynthetic pathways. The invention also provides compounds which can be identified by cloning biosynthetic pathways.

  17. Fast screening method for detection of acyl-HSL-degrading soil isolates

    NARCIS (Netherlands)

    Jafra, S.; Wolf, van der J.M.


    A reliable method was developed for screening of bacteria isolates capable of degrading acyl-HSLs, the signal molecules in quorum-sensing-mediated processes of many Proteobacteria. The microtiter assay was based on the use of a GFP-marked Escherichia coli strain, which fluoresces upon the presence o

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

    DEFF Research Database (Denmark)

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


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

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

    Evaluation of physical, compositional, and digestion characteristics of protozoal glycogen is best performed on a pure substrate in order to avoid interference from other cell components. A method for isolating protozoal glycogen without use of detergents was developed. Rumen inoculum was incubated ...

  20. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues (United States)

    Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...


    Directory of Open Access Journals (Sweden)

    P. Zukowski


    Full Text Available The article presents measurement stand, which is used to measure polarization and depolarization currents, using PDC method, in wet transformer oil-impregnated cellulose isolation. Maximum resolution of measured currents reaches 10-15  A. The accuracy of thermal stabilization, in three-electrode capacitor system, comes to 0,001 °C.

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

    Directory of Open Access Journals (Sweden)

    Sirintip Dangtip


    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.

  3. Simple and efficient methods for enrichment and isolation of endonuclease modified cells.

    Directory of Open Access Journals (Sweden)

    Branden S Moriarity

    Full Text Available The advent of Transcription Activator-Like Effector Nucleases (TALENs, and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO. Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.

  4. An economical and combined method for rapid and efficient isolation of fungal DNA. (United States)

    Lech, T; Syguła-Cholewinska, J; Szostak-Kot, J


    DNA isolation is a crucial step of conducting genetic studies in any organism. However, this process is quite difficult when studying fungi because of the need to damage the fungal cell walls of specific structures. In this study, we developed a method for the rapid and efficient isolation of fungal DNA based on simultaneous mechanical and enzymatic cell wall degradation. There are several typical modifications of the standard phenol-chloroform DNA extraction method. This method can be modified to degrade the fungal cell wall. The first step of the presented DNA extraction included manual homogenization in modified lysis buffer. Next, enzymatic digestion using 2 enzymes was conducted, including lyticase and proteinase K. To carefully select the most favorable conditions, we developed an economical, rapid, and reliable method for fungal DNA extraction that ensures both high efficiency and proper purity, which are essential for further analyses.

  5. A comparative study of two methods for the isolation of human leucocytes for DNA extraction. (United States)

    Lim, L H; Ton, S H; Cheong, S K


    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

  6. Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus (United States)

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


    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. Signal measurement system for intra-body communication using optical isolation method (United States)

    Matsumoto, Kazuki; Katsuyama, Jun; Sugiyama, Ryo; Takizawa, Yasuaki; Ishii, Seita; Shinagawa, Mitsuru; Kado, Yuichi


    In this paper, we describe an induced signal measurement on the human body for developing a high-performance transceiver of an intra-body communication system. It is important to isolate awearable transceiver from an electrical instrument for precise measurement. We have developed a probe system using an optical isolation method including a laser diode, photo-diode, and optical fiber. The probe system can be successfully applied to the precise measurement of a receiving signal power at a wearable transceiver. We verify that the experimental results agree with the simulation results based on our previous channel model of intra-body communication.

  8. Method of selecting vibro-isolation properties of vibration reduction systems

    Energy Technology Data Exchange (ETDEWEB)

    Maciejewski, Igor; Krzyzynski, Tomasz [Koszalin University of Technology, Koszalin (Poland)


    An effective optimization method is proposed in this paper to determine the basic characteristics of non-linear visco-elastic elements used in passive vibration reduction systems. The developed method is examined by performing experimental investigations on the exemplary vibration reduction system, i.e., the passive seat suspension. The shaping of vibro-isolation properties is analyzed for different spectral classes of excitation signals.

  9. Aspergillus nidulans as a platform for discovery and characterization of complex biosynthetic pathways

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere

    andfeed. Secondary metabolites therefore both have a positive and deleterious impact on the human health.The increase in available genome sequences of fungi has revealed that there is a large number of putativesecondary metabolite biosynthetic gene clusters to be discovered and potentially exploited...... of secondary metabolites and 2) Developing A. nidulans as a model systemfor protein production with human-like glycan structure.  The first part of this study resulted in the development of a method for the transfer and expression ofintact biosynthetic gene clusters to A. nidulans to facilitate pathway...... and product discovery. As proof ofconcept the biosynthetic gene cluster for production of the polyketide geodin was identified andtransferred from A. terreus to A. nidulans. The cluster was integrated in a well characterized locus in A.nidulans. Reconstitution of the cluster resulted in the production...

  10. Isolation of entomopathogenic fungi from soils and Ixodes scapularis (Acari: Ixodidae) ticks: prevalence and methods. (United States)

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


    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 litter, and ticks were plated on two types of media; (2) soils were assayed for entomopathogenic fungi using the Galleria bait method; (3) DNA from internal transcribed spacer (ITS) regions of the nuclear ribosomal repeat was extracted from pure cultures obtained from soils, Galleria, and ticks and was amplified and sequenced; and (4) DNA was extracted directly from ticks, amplified, and sequenced. We conclude that (1) ticks encounter potentially entomopathogenic fungi more often in soil than in leaf litter, (2) many species of potentially entomopathogenic fungi found in the soil can readily be cultured, (3) the Galleria bait method is a sufficiently efficient method for isolation of these fungi from soils, and (4) although DNA extraction from ticks was not possible in this study because of small sample size, DNA extraction from fungi isolated from soils and from ticks was successful and provided clean sequences in 100 and 73% of samples, respectively. A combination of the above methods is clearly necessary for optimal characterization of entomopathogenic fungi associated with ticks in the environment.

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

    Directory of Open Access Journals (Sweden)

    Jinyong Kim


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

  12. A Standardized Method of Isolating Adipose-Derived Stem Cells for Clinical Applications. (United States)

    Raposio, Edoardo; Caruana, Giorgia; Petrella, Maira; Bonomini, Sabrina; Grieco, Michele P


    White adipose tissue is the most abundant and accessible source of stem cells in the adult human body. In this paper, we present a standardised and safe method of isolating and maximizing the number of adipose-derived stem cells (ASCs) from conventional liposuction for clinical applications, which was carried out through both mechanical (centrifuge) and enzymatic (collagenase) means. Isolated cells were characterized through flow cytometry assay. Gathered data showed a greater amount (9.06 × 10(5) ASCs from 100 mL of adipose tissue) of isolated ASCs compared to previous protocol, also with high (99%) cell vitality; the procedure we presented is easy and fast (80 minutes), allowing collecting a significative number of mesenchymal stem cells, which can be used for clinical purposes, such as wound healing.

  13. Extending lock-in methods: term isolation detection of nonlinear signals. (United States)

    Jarvis, Thomas W


    We show that components of a nonlinear signal can be measured using phase-sensitive detection at unconventional demodulation frequencies, allowing us to isolate individual terms from the signal. To demonstrate this technique, autocorrelation measurements of an ultrafast pulsed laser were performed using two-photon absorption. In this example, the isolation of individual autocorrelation terms may provide internal consistency checks to improve the precision and accuracy of pulse characterization. More generally, this scheme can be extended to a range of nonlinear measurements. As a demonstration, we analyze a three-photon autocorrelation model, showing that many nonlinear signals can be studied with this method. We anticipate that term isolation detection will find application in a broad range of experiments, such as multidimensional Fourier transform spectroscopy or coherent anti-Stokes Raman spectroscopy.

  14. A comparison of two methods for isolation of Salmonella from poultry litter samples. (United States)

    Read, S C; Irwin, R J; Poppe, C; Harris, J


    Two methods were compared to determine their ability and efficiency in detecting Salmonella in poultry litter samples. Method 1 consisted of pre-enrichment in buffered peptone water (BPW), selective motility enrichment in Modified Semisolid Rappaport Vassiliadis (MSRV) agar, and plating onto MacConkey (MC) agar. Method 2 employed tetrathionate brilliant green (TBG) broth and plating on brilliant green agar with novobiocin (BGAN) and on xylose lysine tergitol 4 (XLT4) agar. Method 1 resulted in a significantly higher isolation rate, was cheaper, and was less labor intensive.

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

    Directory of Open Access Journals (Sweden)

    Baquero-Perez Belinda


    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

  16. A novel method for rapidly isolating microbes that suppress soil-borne phytopathogens (United States)

    Cooper, Sarah; Agnew, Linda; Pereg, Lily


    Seedling establishment faces a large number of challenges related to soil physical properties as well as to fungal root diseases. It is extremely difficult to eliminate fungal pathogens from soils where their populations are established due to the persistent nature of their spores and since fumigation of resident fungi is very ineffective in clay-containing soils. Therefore it is necessary to find ways to overcome disease in areas where the soils are infected with fungal phytopathogens. The phenomenon of disease suppressive soils, where the pathogen is present but no disease observed, suggests that microbial antagonism in the soil may lead to the suppression of the growth of fungal pathogens. There are also cases in the literature where soil microorganisms were isolated that suppress the growth of phytopathogens. Antibiosis is one of the most important mechanisms responsible for fungal antagonism, with some significant antifungal compounds involved including antibiotics, volatile organic compounds, hydrogen cyanide and lytic enzymes. Isolation of pathogen-suppressive microorganisms from the soil is time consuming and tedious. We established a simple method for direct isolation of soil microbes (bacteria and fungi) that suppress fungal phytopathogens as well as procedures for confirmation of disease suppression. We will discuss such methods, which were so far tested with the cotton fungal pathogens Thielaviopsis basicola, Verticillium dahliae and Fusarium oxysporum and Verticillium fungicola. We have isolated a diversity of T. basicola-suppressive fungi and bacteria from two vastly different soil types. Identification of the antagonistic isolates revealed that they are a diverse lot, some belong to groups known to be suppressive of a wide range of fungal pathogens, endorsing the power of this technique to rapidly and directly isolate soil-borne microbes antagonistic to a wide variety of fungal pathogens.

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

    Indian Academy of Sciences (India)

    Gennady V. Pogorelko; Oksana V. Fursova


    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.

  18. 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)


    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.

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

    Directory of Open Access Journals (Sweden)

    Yusuf OZsENSOY


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

  20. Comparative miRNA Analysis of Urine Extracellular Vesicles Isolated through Five Different Methods (United States)

    Royo, Felix; Diwan, Izzuddin; Tackett, Michael R.; Zuñiga, Patricia; Sanchez-Mosquera, Pilar; Loizaga-Iriarte, Ana; Ugalde-Olano, Aitziber; Lacasa, Isabel; Perez, Amparo; Unda, Miguel; Carracedo, Arkaitz; Falcon-Perez, Juan M.


    Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification) and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV)-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis. PMID:27973407

  1. A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

    Institute of Scientific and Technical Information of China (English)

    Tongbao LIU; Jian-ping LU; Xiao-hong LIU; Hang MIN; Fu-cheng LIN


    Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1×106ml-1, the percentages of conidium germination and appressorium formation were (97.98±0.67)% and (97.88±0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 pg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.

  2. Development of a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci. (United States)

    Graham, Ken; Rea, Rosemary; Simpson, Paul; Stack, Helena


    Enterococci show higher proteolytic activities than other lactic acid bacteria and thus have received considerable attention in scientific literature in recent years. Proteolytic enzymes of enterococci have warranted the use of some species as starter, adjuncts or protective cultures and as probiotics, while in some strains they have also been linked with virulence. Consequently, the isolation and identification of proteolytic enterococci is becoming of increasing interest and importance. However, current screening methods for proteolytic enterococci can be time consuming, requiring a two-step procedure which may take up to 96h. This study describes a method, utilising Kanamycin Skim Milk Aesculin Azide (KSMEA) agar, for the isolation of proteolytic enterococci in one-step, thereby significantly reducing screening time. KSMEA combines the selective properties of Kanamycin Aesculin Azide Agar (KAA) with skim milk powder for the detection of proteolytic enterococci. Enterococci produced colonies with a black halo on KSMEA which were accompanied by a zone of clearing in the media when enterococci were proteolytic. KSMEA medium retained the selectivity of KAA, while proteolytic enterococci were easily distinguished from non-proteolytic enterococci when two known strains were propagated on KSMEA. KSMEA also proved effective at isolating and detecting enterococci in raw milk, faeces and soil. Isolates recovered from the screen were confirmed as enterococci using genus-specific primers. Proteolytic enterococci were present in the raw milk sample only and were easily distinguishable from non-proteolytic enterococci and other microorganisms. Therefore, KSMEA provides a rapid, one-step screening method for the isolation of presumptive proteolytic enterococci.

  3. Comparison of the Denka Seiken slide agglutination method to the quellung test for serogrouping of Streptococcus pneumoniae isolates. (United States)

    Shutt, Cheryl K; Samore, Matthew; Carroll, Karen C


    This study compared a slide agglutination test (Denka Seiken, Tokyo, Japan) to the "gold standard" quellung reaction (Pneumotest; Statens Serum Institut, Copenhagen, Denmark) for the serogrouping of pneumococci. Two hundred clinical isolates of Streptococcus pneumoniae were used for the comparison. Each assay was performed according to the manufacturer's instructions. There was an overall agreement of 95.7% between the two methods. Only 4 of 10 isolates of serogroup 22 were detected with the slide agglutination assay. Two isolates that were untypeable by the Pneumotest method were typed as serogroups 6 and 31 by the slide agglutination method. The Pneumotest method was unable to type 22 isolates, and the slide agglutination method was unable to type 16 isolates. The slide agglutination method compares favorably with the Pneumotest method and is easier to perform and to interpret.

  4. Evaluation of different methods for detection of metallo-beta-lactamases in Pseudomonas aeruginosa clinical isolates. (United States)

    Bogiel, Tomasz; Deptuła, Aleksander; Gospodarek, Eugenia


    Metallo-beta-lactamases (MBLs) produced by Pseudomnonas aeruginosa are a serious threat due to their ability to be transmitted between the same as well as different bacterial species. Different methods are applied in the clinical laboratory to detect MBLs. The aim of this study was to compare 4 phenotypic methods and a PCR assay for their ability to detect MBLs in clinical isolates of carbapenem-resistant P. aeruginosa strains. The study embraced a total of 70 carbapenem-resistant P. aeruginosa strains isolated in The Department of Microbiology of Dr. A. Jurasz University Hospital in Bydgoszcz. The highest percentage (42.9%) of the strains were isolated from Intensive Care Unit patients, mainly from urine samples (31.4%). Methods used in this study were: double-disc synergy tests in two combinations: using ceftazidime with 2-mercaptopropionic acid and imipenem with EDTA, differences in inhibition zone diameters between discs with imipenem/EDTA and imipenem, Etest MBL (AB Biodisk) and molecular amplification of bla(IMP) and bla(VIM) genes responsible for producing MBLs, using PCR assay. The lowest percentage (1.4%) of positive results in detection of MBLs was obtained using PCR assay, the highest (72.9%) by double-disc synergy tests with imipenem and EDTA, but the specificity of this method may be low.

  5. 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


    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.

  6. Comparative studies of two methods for miRNA isolation from milk whey. (United States)

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


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

  7. Simple and rapid method for the isolation of forskolin from Coleus forskohlii by charcoal column chromatography. (United States)

    Saleem, A M; Dhasan, P B; Rafiullah, M R M


    A simple, safe, rapid and economical method was developed for the isolation of high-purity forskolin from Coleus forskohlii roots using activated charcoal as an adsorbent in a column. The elution was carried out under reduced pressure to make the process rapid. Activated charcoal acted as a reversed phase adsorbent and allowed elution of forskolin without much impurities. The residue, obtained from the eluate was purified and crystallized using different solvent mixtures to obtain pure forskolin. The forskolin isolated was analyzed and characterized by UV, IR, RP-HPLC, electrospray ionization MS, 1H NMR and 13C NMR. The yield was 0.097% w/w (RSD 5.6%). The purity was 96.9% w/w (RSD 0.3%) as determined by RP-HPLC. The present method enables researchers to produce high-purity forskolin in their labs by using common chemicals.

  8. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms (United States)

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


    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

  9. Natural Product Biosynthetic Diversity and Comparative Genomics of the Cyanobacteria. (United States)

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


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

  10. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms. (United States)

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


    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.

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



    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...

  12. New natural products isolated from Metarhizium robertsii ARSEF 23 by chemical screening and identification of the gene cluster through engineered biosynthesis in Aspergillus nidulans A1145. (United States)

    Kato, Hiroki; Tsunematsu, Yuta; Yamamoto, Tsuyoshi; Namiki, Takuya; Kishimoto, Shinji; Noguchi, Hiroshi; Watanabe, Kenji


    To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an α-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

  13. A rapid method for isolation of total DNA from pathogenic filamentous plant fungi. (United States)

    González-Mendoza, D; Argumedo-Delira, R; Morales-Trejo, A; Pulido-Herrera, A; Cervantes-Díaz, L; Grimaldo-Juarez, O; Alarcón, A


    DNA isolation from some fungal organisms of agronomic importance is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. We have developed a fast DNA isolation protocol for Fusarium oxysporum, which causes fusarium wilt disease in more than 100 plant species, and for Pyrenochaeta terrestris, which causes pink root in onions. This protocol was based on the sodium dodecyl sulfate/phenol method, without beta-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins and co-precipitated polysaccharides. The A(260/280) absorbance ratios of isolated DNA were around 1.9, suggesting that the DNA fraction was pure and may be used for further analysis. Additionally, the A(260/230) values were higher than 1.8, suggesting 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.

  14. 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

  15. Salvinorins D-F, new neoclerodane diterpenoids from Salvia divinorum, and an improved method for the isolation of salvinorin A. (United States)

    Munro, Thomas A; Rizzacasa, Mark A


    Three new neoclerodane diterpenoids, salvinorins D-F (4-6), have been isolated from the leaves of Salvia divinorum. The structures were elucidated by chemical and spectroscopic methods, particularly 1D and 2D NMR. A simplified isolation method using chromatography on activated carbon also gave improved yields of the controlled substance salvinorin A (1) and of salvinorin C (3).

  16. A rapid method for detecting extracellular proteinase activity in Cryptococcus neoformans and a survey of 63 isolates. (United States)

    Ruma-Haynes, P; Brownlee, A G; Sorrell, T C


    A rapid method to detect extracellular proteolytic activity around colonies of Cryptococcus neoformans was developed with tannic acid used to complex with residual protein in a solid medium. A survey was conducted with 32 isolates of C. neoformans var. gattii and 31 isolates of C. neoformans var. neoformans which were cultured on medium containing gelatin as the sole nitrogen source. The annulus of clearing around fungal colonies was > 1.2 mm in 24 (77%) isolates of C. neoformans var. neoformans compared with only 7 (22%) isolates of C. neoformans var. gattii. There was no difference in proteolytic activity between environmental and human clinical isolates of C. neoformans. However, there was a difference between the size of the annulus around animal isolates of C. neoformans var. neoformans and isolates of the same variety from other sources. The annuli around the 14 animal isolates were all >1.2 mm, while 7 (70%) of 10 human clinical isolates and only 3 (43%) of 7 environmental isolates were scored in the high proteinase range. A difference between the genetic types (as characterised by RAPD typing) of C. neoformans var. gattii was also evident with 17 (77%) of 22 VG-I isolates having a small annulus compared with only 1 (17%) of 6 VG-II and VG-III isolates with annuli of similar size. Relatively low proteinase production by C. neoformans var. gattii may reduce local and systemic spread of infection in mammalian hosts.

  17. Development of a method for isolating bovine colostrum mononuclear leukocytes for phenotyping and functional studies. (United States)

    Meganck, Vanessa; Goddeeris, Bruno M; Stuyven, Edith; Piepers, Sofie; Cox, Eric; Opsomer, Geert


    The present study reports a method for isolating bovine colostrum mononuclear cells (CMC) for phenotyping and functional studies. As well as being an important source of immunoglobulins, colostrum also contains leukocytes that may be of greater importance for passive immunity than has previously been thought. Different protocols have been reported for isolating leukocytes from bovine colostrum, although none of these have been validated, and phenotypic analysis of cell populations has not always been performed. In this study, bovine CMC were isolated by density gradient centrifugation. Cell populations were identified by flow cytometry using antibodies against selected bovine cell surface markers and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. The mean cell count of isolated CMC was 3 × 10(4) and 1 × 10(5) per mL colostrum for the samples used in the flow cytometric assay and the proliferation assay, respectively. A mean of 25.4 ± 17.1% CMC were identified as T lymphocytes, 2.9 ± 3.0% as B lymphocytes and 32.7 ± 13.7% as macrophages. In terms of proliferation, the mean counts per minute were 4.3 × 10(3) and 1.8 × 10(4) for cells cultured in medium only or in the presence of concanavalin A, respectively, showing that CMC are viable and capable of responding to mitogen stimulation. Isolation of CMC and the subsequent phenotypic analysis of the different subpopulations were repeatable, with agreement indices varying between 0.5 and 1.0. Agreement indices for the proliferation assay were estimated at 0.8.

  18. Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma

    Directory of Open Access Journals (Sweden)

    Ferreira Ricardo B


    Full Text Available Abstract Background RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible with downstream applications such as gene expression. Findings In this work, a comparative analysis of the RNA quality obtained from SK-N-MC cells was performed using six commonly used RNA isolation kits: two phenol-based kits and four non-phenol based kits. The non-phenol based kits tested AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini, EasySpin and Ilustra RNAspin Mini RNA Isolation, all performed well and resulted in the isolation of high quality RNA, as evaluated by A260/A280. The RNA extracted with AxyPrep Multisource Total RNA Miniprep, RNeasy® Mini and EasySpin provided the highest RNA yields. In particular, the RNA isolated by AxyPrep Multisource Total RNA Miniprep Kit did not show any detectable genomic DNA contamination even without previous DNase treatment or after RNA direct PCR amplification using universal 18S primers. Conclusions The RNA extracted from SK-N-MC cells with AxyPrep Multisource Total RNA Miniprep Kit was superior with respect to the RNA quality and concentration. This kit does not use aggressive organic solvents and RNA free of genomic DNA was isolated without the need for DNase treatment.

  19. 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


    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 ( -- 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.

  20. Modified isolation method of Arcobacter spp. from different environmental and food samples. (United States)

    Šilha, David; Šilhová-Hrušková, Lucie; Vytřasová, Jarmila


    This study provides information on the occurrence of Arcobacter in several types of water and food products of animal origin in the Czech Republic. We processed 190 samples using the modified method, and the occurrence of Arcobacter spp. was confirmed in 36.8 % of these. This total incidence consisted of Arcobacter butzleri (27.3 %), Arcobacter cryaerophilus (8.4 %) and Arcobacter skirrowii (1.1 %). We newly described the common presence of Arcobacter spp. in sewage water in the Czech Republic that is released into waterways after processing in water treatment plants (86.7 %). All the acquired isolates were subject to detailed confirmation with subsequent species classification using multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we used a modification of a method using passive filtration of an enriched sample, which could be suitable for the isolation of Arcobacter, especially in combination with Campylobacter selective agar chromogenic medium. Our studies have shown this agar to be quite suited to the isolation of Arcobacter and that it can be an appropriate instrument for accelerating culture diagnostics.

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


    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.

  2. Technical note: A method for isolating glycogen granules from ruminal protozoa for further characterization. (United States)

    Hall, Mary Beth


    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.

  3. [Improvement of the method of isolation of hydrogen-forming bacteria of Clostridium genus]. (United States)

    Pritula, I R; Tashirev, A B


    The method of isolation and quantitative account of pure cultures of obligate anaerobic hydrogen-forming clostridia is improved. A strain of hydrogen-forming bacteria Clostridium sp. BY-11 has been isolated from the association of sporulating bacteria. Quantitative indices of hydrogen synthesis and starch fermentation have been determined when growing the strain in the liquid medium. Concentration of H2 in the gas phase was 49%, microorganisms synthesized 128 1 of H2 from 1 kg of starch, the mass of starch decreased 7 times for 6 days. The mentioned indices for hydrogen synthesis and starch fermentation and for other organic model substrates in the future are the basis for creating the industrial biotechnology for production of hydrogen as the energy carrier under disposal of ecologically dangerous solid food waste.

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Megan Coomer


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

  6. 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)


    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.

  7. Minimum Information about a Biosynthetic Gene cluster : commentary

    NARCIS (Netherlands)

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


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

  8. Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells. (United States)

    Kumar, R; Ahlawat, S P S; Sharma, M; Verma, O P; Sai Kumar, G; Taru Sharma, G


    The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2-5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.

  9. Genetic Diversity among Ciprofloxacin Resistant Enterococcus faecalis Isolated from Clinical Specimens with Pulsed-Field Gel Electrophoresis (PFGE Method

    Directory of Open Access Journals (Sweden)

    Moinian M


    Full Text Available Abstract Background and objective: Resistance to ciprofloxacin among Enterococcus faecalis (E.f isolates especially in UTI makes difficulties for treatment. In this study, the genetic diversity using PFGE method and detection of resistance genes including parC, gyrA , gyrB and parE among ciprofloxacin resistant E.f isolated from clinical specimens, are determined. Materials and methods: A total of 384 entreococcal isolates were collected from 6 hospitals and 3 private laboratories in Tehran and 50 ciprofloxacin resistant E.f isolates were obtained. Identification of species and resistance genes were done by PCR method. Antimicrobial and minimum inhibitory concentration (MICs tests were assayed with standard methods and finally genotyping was accomplished using PFGE method. Results: The range of ciprofloxacin MICs was 16 to 512 µg/ml. All of these isolates contained parC, 98 % gyrA , gyrB and 80 % parE genes. PFGE analysis, grouped 50 strains in 11 common types and 7 single types. The P4, P9 and P10 genotypes were shared between hospital and community isolates. Conclusion: According to these results the E.f isolates showed high clonal diversity. Because of the ciprofloxacin high MICs level among common pulsotypes we concluded that they have various distributions which may be due to highly transmission of resistant genes among enterococci. Indeed the colonized patients with these resistant isolates are reservoir for releasing of the resistant genes to community which requires more surveillance programs.

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

    Directory of Open Access Journals (Sweden)

    Alyssa M Walterson

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Dionicia Gloria León-Martínez


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

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

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

  13. 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


    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.

  14. Association between Pseudonocardia symbionts and Atta leaf-cutting ants suggested by improved isolation methods. (United States)

    Marsh, Sarah E; Poulsen, Michael; Gorosito, Norma B; Pinto-Tomás, Adrián; Masiulionis, Virginia E; Currie, Cameron R


    Fungus-growing ants associate with multiple symbiotic microbes, including Actinobacteria for production of antibiotics. The best studied of these bacteria are within the genus Pseudonocardia, which in most fungus-growing ants are conspicuously visible on the external cuticle of workers. However, given that fungus-growing ants in the genus Atta do not carry visible Actinobacteria on their cuticle, it is unclear if this genus engages in the symbiosis with Pseudonocardia. Here we explore whether improving culturing techniques can allow for successful isolation of Pseudonocardia from Atta cephalotes leaf-cutting ants. We obtained Pseudonocardia from 9 of 11 isolation method/colony component combinations from all 5 colonies intensively sampled. The most efficient technique was bead-beating workers in phosphate buffer solution, then plating the suspension on carboxymethylcellulose medium. Placing these strains in a fungus-growing ant-associated Pseudonocardia phylogeny revealed that while some strains grouped with clades of Pseudonocardia associated with other genera of fungus-growing ants, a large portion of the isolates fell into two novel phylogenetic clades previously not identified from this ant-microbe symbiosis. Our findings suggest that Pseudonocardia may be associated with Atta fungus-growing ants, potentially internalized, and that localizing the symbiont and exploring its role is necessary to shed further light on the association.

  15. "Aqua-space", a new headspace method for isolation of natural floral aromas using humidified air as a carrier gas. (United States)

    Ishikawa, Masashi; Honda, Tsutomu; Fujita, Akira; Kurobayashi, Yoshiko; Kitahara, Takeshi


    A new method called "Aqua-space" was developed for the isolation of the natural fragrances of plants. Living flowers were enclosed in a space under simulated natural conditions, and humidified air was pumped into the space as a fragrance carrier. In a comparison among three isolation methods, i.e., Aqua-space, headspace, and solvent extraction, the Aqua-space method proved to be the most effective in retaining natural fragrances with abundant oxygenated components key to floral fragrances.

  16. 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


    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.

  17. 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

  18. Application of a radiometric method for evaluation of loss of salicylic acid during isolation from biologic material. (United States)

    Ostrowski, A


    A radiometric method for evaluation of loss of salicylic acid in the process of isolation from biologic material is described. According to this study the mean loss during the total process of isolation amounts to 33.59%, the specific values being 19.47% during protein precipitation, 10.68% during extraction, and 3.44% during evaporation of solvent.

  19. Effects of Phytophthora cinnamomi isolate, inoculum delivery method, flood, and drought on vigor, disease severity and mortality of blueberry plants (United States)

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

  20. Method for isolating chromosomal DNA in preparation for hybridization in suspension (United States)

    Lucas, Joe N.


    A method is provided for detecting nucleic acid sequence aberrations using two immobilization steps. According to the method, a nucleic acid sequence aberration is detected by detecting nucleic acid sequences having both a first nucleic acid sequence type (e.g., from a first chromosome) and a second nucleic acid sequence type (e.g., from a second chromosome), the presence of the first and the second nucleic acid sequence type on the same nucleic acid sequence indicating the presence of a nucleic acid sequence aberration. In the method, immobilization of a first hybridization probe is used to isolate a first set of nucleic acids in the sample which contain the first nucleic acid sequence type. Immobilization of a second hybridization probe is then used to isolate a second set of nucleic acids from within the first set of nucleic acids which contain the second nucleic acid sequence type. The second set of nucleic acids are then detected, their presence indicating the presence of a nucleic acid sequence aberration. Chromosomal DNA in a sample containing cell debris is prepared for hybridization in suspension by treating the mixture with RNase. The treated DNA can also be fixed prior to hybridization.

  1. 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


    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.

  2. [In vitro susceptibility of isolates of Paracoccidioides spp complex to systemic antifungals using the microdilution method]. (United States)

    Cermehol, Julman R; Alvarado, Primavera; Mendoza, Mireya; Herndndez, Isabel; Cuestal, De


    Broth microdilution, the reference method recommended by the Clinical Laboratory Standards Institute (CLSI), is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this work, in vitro susceptibility of the Paracoccidioides complex (n=19) to systemic antifungals: amphotericin B, 5-flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole and caspofungin, was evaluated using the microdilution method (Document M27-A3, M27-S3), with some modifications such as: culture time in Sabouraud dextrose agar (7-10 days), RPMI 1640 medium supplemented with 2% glucose and the incubation time (7, 8 and 18 days). The sensitivity in vitro was variable; the majority of Paracoccidioides isolates was susceptible to ketoconazol (73.7%), followed by voriconazole (68.4%), itraconazole (63.1%), amphotericin B (52.6%), fluconazole (47.4%), 5-flucytosine (42.1%) and caspofungin (5%). The overall resistance was mainly to caspofungin (94.7%), followed by 5-flucytosine (52.6%) and amphotericin B (47.4%). Fifty-three percent of the isolates were susceptible-dose dependent to fluconazole followed by itraconazole (15.7%) and 5-fluorocytosine (5.3%). Amphotericin B, itraconazole and voriconazole were the most potent antifungal drugs against Paracoccidioides spp (CMI: 0.03-1 microg/mL). Based on these results, we tentatively propose a microdilution assay protocol for susceptibility testing of Paracoccidioides spp to antifungal drugs. This method may be clinically useful to predict resistance, even though further studies are needed.

  3. 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


    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.

  4. To a Method of Polarization-Depolarization Currents for Diagnosis of Dielectric Isolation (United States)

    Ambrozevich, S. A.; Sibatov, R. T.; Uchaikin, D. V.; Morozova, E. V.


    Fractional derivative formalism is proposed as the mathematical foundation of the polarization-depolarization current method for the diagnosis of dielectric isolation. Physical basis of the new approach is the observed deviation of the long-term relaxation from the Debye exponential law. We found that this behavior is consistent with the solution of the fractional differential equation: exponential behavior turns into the power dependence in the long-time asymptotics, and this part of the relaxation curve is more sensitive to the material state. The results of calculations for the polarization-depolarization currents in an oil-paper capacitor are in agreement with the specially performed experiments.

  5. [Effective method of isolating M4-lactate dehydrogenase from rat liver]. (United States)

    Gorbach, Z V; Maglysh, S S; Konovalenko, O V


    Lactate dehydrogenase M4-isoform in the homogeneous state was isolated from the rat liver by successive application of sulphate-ammonium fractionation, phosphocellulose ion-exchange chromatography with high-affinity elution of 1 mM NADH and subsequent hydroxyl apatite fractionation. The method permits obtaining the preparation amounts of the enzymic protein with yield 37.5%, specific activity 386.8 units per 1 mg of protein. It is established that 1 mM NAD+, 10 mM pyruvate and 100 mM lactate are also effective as agents of the selective enzyme elution.

  6. A generic plant RNA isolation method suitable for RNA-Seq and suppression subtractive hybridization. (United States)

    Zhu, Y Q; Wu, W J; Xiao, H W; Chen, H B; Zheng, Y; Zhang, Y J; Wang, H X; Huang, L Q


    A recently developed revolutionary approach to transcriptomics, RNA-Seq, and suppression subtractive hybridization are powerful tools for gene expression research. However, currently, the difficulty of isolating high-quality RNAs from plant tissues bearing abundant complex polysaccharides, polyphenolics, and secondary metabolites is a serious problem that not only limits the application of these technologies but also hinders studies dealing with RNA in general. We have developed a consistent protocol to prepare highly intact and pure RNAs from tissues of a variety of field-grown plant species, with high yields, in 2 to 3 h. Additionally, this method can be readily applied to mammalian, yeast, and bacterial cells.

  7. Measurement of the Ar diffusion coefficient in graphite at high temperature by the ISOL method

    Energy Technology Data Exchange (ETDEWEB)

    Eleon, C. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France); Jardin, P. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France)], E-mail:; Thomas, J.C.; Saint-Laurent, M.-G.; Huet-Equilbec, C.; Alves Conde, R. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France); Angelique, J.C. [Laboratoire de Physique Subatomique et de Cosmologie, 38026 Grenoble (France); Laboratoire de Physique Corpusculaire, ISMRA, 14050 Caen (France); Boilley, D.; Cornell, J.; Dubois, M. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France); Franberg, H. [Paul Scherrer Institute, 5232 Villigen PSI (Switzerland); ISOLDE, CERN, 1211 Geneve 23 (Switzerland); Gaubert, G.; Jacquot, B. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France); Koester, U. [ISOLDE, CERN, 1211 Geneve 23 (Switzerland); Institut Laue Langevin, 38042 Grenoble (France); Leroy, R. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France); Maunoury, L. [Centre Interdisciplinaire de Recherche Ion Laser, 14070 Caen (France); Orr, N. [Laboratoire de Physique Corpusculaire, ISMRA, 14050 Caen (France); Pacquet, J.Y.; Pellemoine, F.; Stodel, C. [Grand Accelerateur National d' Ions Lourds, CEA/DSM CNRS/IN2P3, 14076 Caen (France)] (and others)


    This work has been carried out at GANIL within the ambit of the TARGISOL European collaboration which aims to study the relevant variables governing the release of radioactive elements from targets in an ISOL system. This work shows how it has been possible to extract diffusion coefficients for {sup 35}Ar atoms diffusing out of graphite targets from release time measurements by using an analytic description of the release times. The diffusion coefficients and efficiencies are presented and compared with results obtained using a 'continuous' method.

  8. Methods for the isolation of genes encoding novel PHB cycle enzymes from complex microbial communities. (United States)

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


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

  9. [Computer simulation of the isolated lesion of tibiofibular an syndesmosis using the finite element method]. (United States)

    Kozień, Marek S; Lorkowski, Jacek; Szczurek, Sławomir; Hładki, Waldemar; Trybus, Marek


    The aim of this study was to construct a computed simulation of an isolated lesion of tibiofibular syndesmosis on typical clinical range of value. The analysis was made using the method of finite elements with a simplified plain model of a bone and assuming material of bone and ankle joint as isotropic and homogeneous. The distraction processes were modelled by external generalized forces. The computed programme ANSYS was used. For evaluation obtained was the computed image of changes of anatomy in relation to forces.

  10. Isolation and catalytic competence of different animal liver microsomal fractions prepared by calcium-aggregation method

    Directory of Open Access Journals (Sweden)

    Walawalkar Pradnya


    Full Text Available Rat, mouse, rabbit, and guinea pig liver microsomes were prepared, using the calcium-aggregation method. The mean specific spectral cytochrome P450 content obtained for rat, mouse, rabbit, and guinea pig liver microsomes were 0.547, 0.394, 0.677, and 0.378 nmol cytochrome P450/mg protein, respectively. All the microsomal samples showed the ability to biotransform p-nitrophenol to p-nitrocatechol. The mean rate of formation of p-nitrocatechol by rat, mouse, rabbit, and guinea pig liver microsomes was 0.272, 0.247, 0.497 and 0.424 nmole/min/nmole cytochrome 450, respectively. This method circumvents the need of an ultracentrifuge for isolation of microsomes, by the conventional differential centrifugation method.

  11. Isolation and Identification of Listeria monocytogenes in Processed Meat by a Combined Cultural-molecular Method

    Directory of Open Access Journals (Sweden)

    Angela Ingianni


    Full Text Available The isolation and identification of Listeria monocytogenes in processed meat samples by a combined cultural-molecular method is described. It allows the identification of Listeria strains by means of a hybridization technique with a specific DNA probe directed to the listerial internalin gene. The specificity of this method was found to be 100% and sensitivity was as low as 1 CFU/2.5 g of food sample. A total of 278 meat samples were tested in comparison with PCR and conventional cultural assays. A total of 42 (15.4% L. monocytogenes were detected. PCR analysis gave 3 false negative results and culture failed to detect the Listeria in 5 cases. With this cultural-molecular method the identification and quantitative detection of L. monocytogenes were achieved within 36 hours and no false positive or negative tests were obtained, thus fitting most food industry requirements.

  12. A novel 4-hydroxycoumarin biosynthetic pathway. (United States)

    Liu, Benye; Raeth, Torben; Beuerle, Till; Beerhues, Ludger


    Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.

  13. Methods of detection and typing of methicillin resistant Staphylococcus aureus isolated from animals

    Directory of Open Access Journals (Sweden)

    Radosavljević V.


    Full Text Available In this work there was evaluated the method of detection of methicillin resistant Staphylococcus aureus (MRSA by using two molecular and three phenotypic tests in investigation procedure of 70 strains of S.aureus isolated from animals. Recent findings of the new mecA homologue, mecALGA251, minimise the significance of mecA gene presence detection as a confirmation method of methicillin resistant Staphylococcus aureus identification. For this reason, along with multiplex PCR set of primers(165rDNK, nuc, mecA for detection mecA gene, there was also used multiplex PCR set of primers (spa, mecA, pvl, mecALGA251 for differentiation mecALGA251 from mecA, with simultaneous detection of luk-PV and spa gene fragments. In all 70 investigated isolates there was detected the presence of specific 16 SrDNK fragment and nuc gene which encodes a thermostable S. aureus nuclease, while in 5 out of 70 S. aureus isolates, there was proven mecA gene presence using two multiplex PCR tests. In the investigated strains there was determined neither mecC (mecALGA251gene presence, nor Panton Valentine Leukocidin encoding gene. By application cefoxitin disk-diffusion, latex-agglutination and two multiplex PCR tests, the identical results in identification 5 methicillin resistant out of 70 investigated S. aureus strains were obtained. In our investigation there was determined a complete correlation between the results of phenotypic and genotypic identification of methicillin resistant S. aureus. [Projekat Ministarstva nauke Republike Srbije, br. TR 31079

  14. Diversity of Culturable Thermophilic Actinobacteria in Hot Springs in Tengchong, China and Studies of their Biosynthetic Gene Profiles. (United States)

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


    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


    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine


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

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

    Directory of Open Access Journals (Sweden)

    Desiana Radithia


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

  17. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Vincitorio Massimo


    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.

  19. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells. (United States)

    Corti, Davide; Lanzavecchia, Antonio


    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  20. A rapid method for isolation of piperine from the fruits of Piper nigrum Linn. (United States)

    Kanaki, Niranjan; Dave, Mansi; Padh, Harish; Rajani, Mandapati


    A simple, rapid and efficient method has been developed for the isolation of piperine from the fruits of Piper nigrum. The method involves extraction of the fruit powder with glacial acetic acid, from which piperine is partitioned into chloroform and subsequently crystallized. The identity of the compound was confirmed by its melting point, comparison of UV, IR, and mass spectral data with those from a reference standard, and co-chromatography with the reference standard using thin-layer chromatography (TLC). The purity of the compound was ascertained by TLC, by recording UV absorption spectra at the start, middle, and end positions of the spot on the plate, and by differential scanning calorimetry (DSC).

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

    DEFF Research Database (Denmark)

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


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

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

    DEFF Research Database (Denmark)

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


    on a porous membrane was superior to the other methods applied [P cultivated cells. CM had similar viability supporting effects to FCS. Other supplements had no effects......Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy...... and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different...

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


    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.

  4. Revision of the stereochemistry of elisabethatriene, a putative biosynthetic intermediate of pseudopterosins. (United States)

    Nasuda, Masayuki; Ohmori, Miho; Ohyama, Kiyoshi; Fujimoto, Yoshinori


    In the past, we have questioned the accuracy of the stereochemistry of elisabethatriene, a putative biosynthetic intermediate of pseudopterosins, in light of the configuration of elisabethatrienol isolated from Pseudopterogorgia elisabethae, which was represented as 1S,4R,9S,11S. We have reinvestigated the stereochemistry of elisabethatriene. Elisabethatriene with the reported 1S,4R,9R,11S configuration was synthesized starting from (-)-isopulegol in its enantiomeric form. The (1)H- and (13)C-NMR data of the synthesized compound differed from those reported for elisabethatriene. In addition to the fact that elisabethatriene is converted into pseudopterosins, this finding has allowed us to propose that elisabethatriene should have the 1S,4R,9S,11S stereochemistry, which is identical to that of elisabethatrienol.

  5. Metagenomic approaches for exploiting uncultivated bacteria as a resource for novel biosynthetic enzymology. (United States)

    Wilson, Micheal C; Piel, Jörn


    Most biologically active microbial natural products are known from strains that can be isolated and cultivated in the laboratory. However, the genomics era has revealed that cultured bacteria represent a mere fraction of total estimated bacterial biodiversity. With the development of community genomics, termed metagenomics, the uncultivated majority became accessible for functional analysis. Through metagenomic studies, novel biocatalysts and biosynthetic pathways are being discovered at a pace previously not possible using traditional molecular biology techniques. Additionally, the study of uncultivated bacteria has provided valuable insights into previously overlooked biocatalysts from cultured strains. This perspective highlights recent discoveries from metagenomics of uncultivated bacteria and discusses the impact of those findings on the field of natural products.

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

    DEFF Research Database (Denmark)

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


    production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established......BACKGROUND: Fungal polyketides include commercially important pharmaceuticals and food additives, e.g. the cholesterol-lowering statins and the red and orange monascus pigments. Presently, production relies on isolation of the compounds from the natural producers, and systems for heterologous....... CONCLUSIONS: The reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction...

  7. Didemnin Biosynthetic Gene Cluster In Tistrella Mobilis

    KAUST Repository

    Qian, Pei-Yuan


    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.

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

    Directory of Open Access Journals (Sweden)

    André Bellin Mariano


    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.

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

    Directory of Open Access Journals (Sweden)

    Teresa eDocimo


    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

  10. Ethylene biosynthetic genes are differentially expressed during carnation (Dianthus caryophyllus L.) flower senescence. (United States)

    ten Have, A; Woltering, E J


    Ethylene production and expression patterns of an 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (CARAO1) and of two ACC synthase (EC genes (CARACC3 and CARAS1) were studied in floral organs of cut carnation flowers (Dianthus caryophyllus L.) cv. White Sim. During the vase life and after treatment of fresh flowers with ethylene, production of ethylene and expression of ethylene biosynthetic genes first started in the ovary followed by the styles and the petals. ACC oxidase was expressed in all the floral organs whereas, during the vase life, tissue-specific expression of the two ACC synthase genes was observed. After treatment with a high ethylene concentration, tissue specificity of the two ACC synthase genes was lost and only a temporal difference in expression remained. In styles, poor correlation between ethylene production and ACC synthase (CARAS1) gene expression was observed suggesting that either activity is regulated at the translational level or that the CARAS1 gene product requires an additional factor for activity. Isolated petals showed no increase in ethylene production and expression of ethylene biosynthetic genes when excised from the flower before the increase in petal ethylene production (before day 7); showed rapid cessation of ethylene production and gene expression when excised during the early phase of petal ethylene production (day 7) and showed a pattern of ethylene production and gene expression similar to the pattern observed in the attached petals when isolated at day 8. The interorgan regulation of gene expression and ethylene as a signal molecule in flower senescence are discussed.

  11. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

    Directory of Open Access Journals (Sweden)

    Patricia Müller-Moulé


    Full Text Available Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

  12. YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance (United States)

    Müller-Moulé, Patricia; Nozue, Kazunari; Pytlak, Melissa L.; Palmer, Christine M.; Covington, Michael F.; Wallace, Andreah D.; Harmer, Stacey L.


    Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance. PMID:27761349

  13. 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.

  14. Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes. (United States)

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


    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.

  15. Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies (United States)

    Meyer, Anke; Paroni, Federico; Günther, Kathrin; Dharmadhikari, Gitanjali; Ahrens, Wolfgang; Kelm, Sørge; Maedler, Kathrin


    Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or

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

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Haqqani Arsalan S


    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

  18. Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation. (United States)

    Yamashita, Takuma; Takahashi, Yuki; Nishikawa, Makiya; Takakura, Yoshinobu


    Exosomes, which are expected to be delivery systems for biomolecules such as nucleic acids, are collected by several methods. However, the effect of exosome isolation methods on the characteristics of exosomes as drug carriers, such as recovery efficiency after sterile filtration and pharmacokinetics, has not been investigated despite the importance of these characteristics for the development of exosome-based delivery systems. In the present study, exosomes collected from murine melanoma B16-BL6 cells by several methods were compared with respect to dispersibility, recovery rate after filtering, and clearance from the blood circulation in mice. The exosomes were collected by three ultracentrifugation-based methods: simple ultracentrifugation/pelleting (pelleting method), ultracentrifugation with an iodixanol cushion (cushion method), and ultracentrifugation on an iodixanol density gradient (gradient method). The isolation methods had little effect on the particle number of exosomes. In contrast, transmission electron microscopy observation and size distribution measurement using tunable resistive pulse sensing indicated that the exosomes of the gradient method were more dispersed than the others. The exosomes were labeled with Gaussia luciferase and intravenously injected into mice. Clearance of injected exosomes from the blood circulation did not significantly change with isolation methods. When the exosomes were filtered using a 0.2-μm filter, the recovery rate was 82% for the exosomes of the gradient method, whereas it was less than 50% for the others. These results indicate that the exosome isolation method markedly affects the dispersibility and filtration efficiency of the exosomes.

  19. New biosynthetic pathway for pink pigments from uncultured oceanic viruses. (United States)

    Ledermann, Benjamin; Béjà, Oded; Frankenberg-Dinkel, Nicole


    The pink open-chain tetrapyrrole pigment phycoerythrobilin (PEB) is employed by marine cyanobacteria, red algae and cryptophytes as a light-harvesting chromophore in phycobiliproteins. Genes encoding biosynthesis proteins for PEB have also been discovered in cyanophages, viruses that infect cyanobacteria, and mimic host pigment biosynthesis with the exception of PebS which combines the enzymatic activities of two host enzymes. In this study, we have identified novel members of the PEB biosynthetic enzyme families, heme oxygenases and ferredoxin-dependent bilin reductases. Encoding genes were found in metagenomic datasets and could be traced back to bacteriophage but not cyanophage origin. While the heme oxygenase exhibited standard activity, a new bilin reductase with highest homology to the teal pigment producing enzyme PcyA revealed PEB biosynthetic activity. Although PcyX possesses PebS-like activity both enzymes share only 9% sequence identity and likely catalyze the reaction via two independent mechanisms. Our data point towards the presence of phycobilin biosynthetic genes in phages that probably infect alphaproteobacteria and, therefore, further support a role of phycobilins outside oxygenic phototrophs.

  20. Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes. (United States)

    Kriechbaumer, Verena; Seo, Hyesu; Park, Woong June; Hawes, Chris


    Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.


    Directory of Open Access Journals (Sweden)

    Shankar Katekhaye


    Full Text Available Simple, rapid and specific isolation and HPLC method has been develop for filixic acid PBP in Dryopteris filix-mas extract. HPLC analysis was performed on C18 column using a 90:10 (v/v mixtures of acetonitrile and methanol as isocratic mobile phase at a flow rate 1.0 ml/min. UV detection was at 254 nm for filixic acid PBP. Filixic acid shown retention time at 4.02 min. Method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International Conference on Harmonisation guidelines. Validation studies revealed that the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficient (r2 > 0.993 was obtained for calibration plots in the range tested. The limit of detection was 2.25 µg/ml and limit of quantification was 7.53 µg/ml for filixic acid PBP. Intra and inter-day relative standard deviation of precision was less than 1.50 %. Recovery was between 94.57 and 101.05 % filixic acid. The method can be successfully used for quantitative analysis of filixic acid PBP in D. filix-mas for day-to-day studies.

  2. Use of a Novel Teaching Method to Increase Knowledge and Adherence to Isolation Procedures. (United States)

    Anderson, Nancy; Johnson, Diane; Wendt, Lindsey


    Adherence to infection prevention precautions is a challenge in many hospitals. The purpose of this study was to determine if an isolation educational program, which included visual demonstrations of cross-contamination during breaks in isolation procedures, increased nursing staff knowledge of and adherence to isolation procedures.

  3. A unique method for the isolation of nasal olfactory stem cells in living rats. (United States)

    Stamegna, Jean-Claude; Girard, Stéphane D; Veron, Antoine; Sicard, Gilles; Khrestchatisky, Michel; Feron, François; Roman, François S


    Stem cells are attractive tools to develop new therapeutic strategies for a variety of disorders. While ethical and technical issues, associated with embryonic, fetal and neural stem cells, limit the translation to clinical applications, the nasal stem cells identified in the human olfactory mucosa stand as a promising candidate for stem cell-based therapies. Located in the back of the nose, this multipotent stem cell type is readily accessible in humans, a feature that makes these cells highly suitable for the development of autologous cell-based therapies. However, preclinical studies based on autologous transplantation of rodent olfactory stem cells are impeded because of the narrow opening of the nasal cavity. In this study, we report the development of a unique method permitting to quickly and safely biopsy olfactory mucosa in rats. Using this newly developed technique, rat stem cells expressing the stem cell marker Nestin were successfully isolated without requiring the sacrifice of the donor animal. As an evidence of the self-renewal capacity of the isolated cells, several millions of rat cells were amplified from a single biopsy within four weeks. Using an olfactory discrimination test, we additionally showed that this novel biopsy method does not affect the sense of smell and the learning and memory abilities of the operated animals. This study describes for the first time a methodology allowing the derivation of rat nasal cells in a way that is suitable for studying the effects of autologous transplantation of any cell type present in the olfactory mucosa in a wide variety of rat models.

  4. Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples

    Institute of Scientific and Technical Information of China (English)

    Heng-Xiang; Wang; Zhi-Yong; Li; Zhi-Kun; Guo; Zi-Kuan; Guo


    AIM:To establish an easily-handled method to isolate mesenchymal stem cells(MSCs) from coagulated human bone marrow samples. METHODS: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated,treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 ℃ for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast(CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents.RESULTS: The average CFU-F number of urokinasetreated samples(16.85 ± 11.77/106) was comparable to that of un-coagulated control samples(20.22 ± 10.65/106,P = 0.293),which was significantly higher than those of mechanically-cut clots(6.5 ± 5.32/106,P < 0.01) and untreated clots(1.95 ± 1.86/106,P < 0.01). The CFU-F numbers decreased after samples were stored,but those of control and urokinase-treated clots remained higher than the other two groups. Consistently,the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots.The attached cells were fibroblast-like in morphology and homogenously positive for CD44,CD73 and CD90,and negative for CD31 and CD45. Also,they could be induced to differentiate into osteoblasts and adipocytes in vitro. CONCLUSION: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

  5. A New Isolation and Evaluation Method for Marine-Derived Yeast spp. with Potential Applications in Industrial Biotechnology. (United States)

    Zaky, Abdelrahman Saleh; Greetham, Darren; Louis, Edward J; Tucker, Greg A; Du, Chenyu


    Yeasts that are present in marine environments have evolved to survive hostile environments that are characterized by high exogenous salt content, high concentrations of inhibitory compounds, and low soluble carbon and nitrogen levels. Therefore, yeasts isolated from marine environments could have interesting characteristics for industrial applications. However, the application of marine yeast in research or industry is currently very limited owing to the lack of a suitable isolation method. Current methods for isolation suffer from fungal interference and/or low number of yeast isolates. In this paper, an efficient and non-laborious isolation method has been developed and successfully isolated large numbers of yeasts without bacterial or fungal growth. The new method includes a three-cycle enrichment step followed by an isolation step and a confirmation step. Using this method, 116 marine yeast strains were isolated from 14 marine samples collected in the UK, Egypt, and the USA. These strains were further evaluated for the utilization of fermentable sugars (glucose, xylose, mannitol, and galactose) using a phenotypic microarray assay. Seventeen strains with higher sugar utilization capacity than the reference terrestrial yeast Saccharomyces cerevisiae NCYC 2592 were selected for identification by sequencing of the ITS and D1/D2 domains. These strains belonged to six species: S. cerevisiae, Candida tropicalis, Candida viswanathii, Wickerhamomyces anomalus, Candida glabrata, and Pichia kudriavzevii. The ability of these strains for improved sugar utilization using seawater-based media was confirmed and, therefore, they could potentially be utilized in fermentations using marine biomass in seawater media, particularly for the production of bioethanol and other biochemical products.

  6. Equine preantral follicles obtained via the Biopsy Pick-Up method: histological evaluation and validation of a mechanical isolation technique. (United States)

    Haag, K T; Magalhães-Padilha, D M; Fonseca, G R; Wischral, A; Gastal, M O; King, S S; Jones, K L; Figueiredo, J R; Gastal, E L


    The aims of this study in mares were to: (1) compare preantral follicle parameters between in vitro Biopsy Pick-Up (BPU) and scalpel blade collection methods and between histological and mechanical isolation processing (experiment 1); (2) histologically evaluate preantral follicles (experiment 2); and (3) compare histological analysis with a previously established mechanical isolation technique using a tissue chopper (experiment 3) for ovarian cortical fragments obtained in vivo using a BPU instrument. In experiment 1, preantral follicles were analyzed (N = 220; 90% primordial and 10% primary). Proportions of primordial and primary follicles did not differ (P > 0.05) between tissue collection (BPU vs. scalpel blade dissection) or processing (mechanical isolation vs. histology) methods. Follicle viability and morphology rates were similar (P > 0.05) between tissue collection methods, but mechanical isolation produced more (P 0.05) by processing methods. In conclusion, most parameters evaluated for preantral follicles were similar between histological and tissue chopper processing techniques; hence, mechanical isolation efficiently dissociated equine preantral follicles from the ovarian cortex. Therefore, the tissue chopper could be used to isolate large numbers of morphologically normal equine preantral follicles for cryopreservation and/or in vitro culture.

  7. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells. (United States)

    Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li


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

  8. Anaerobic High-Throughput Cultivation Method for Isolation of Thermophiles Using Biomass-Derived Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton-Brehm, Scott [ORNL; Vishnivetskaya, Tatiana A [ORNL; Allman, Steve L [ORNL; Mielenz, Jonathan R [ORNL; Elkins, James G [ORNL


    Flow cytometry (FCM) techniques have been developed for sorting mesophilic organisms, but the difficulty increases if the target microbes are thermophilic anaerobes. We demonstrate a reliable, high-throughput method of screening thermophilic anaerobic organisms using FCM and 96-well plates for growth on biomass-relevant substrates. The method was tested using the cellulolytic thermophiles Clostridium ther- mocellum (Topt = 55 C), Caldicellulosiruptor obsidiansis (Topt = 78 C) and the fermentative hyperthermo- philes, Pyrococcus furiosus (Topt = 100 C) and Thermotoga maritima (Topt = 80 C). Multi-well plates were incubated at various temperatures for approximately 72 120 h and then tested for growth. Positive growth resulting from single cells sorted into individual wells containing an anaerobic medium was verified by OD600. Depending on the growth substrate, up to 80 % of the wells contained viable cultures, which could be transferred to fresh media. This method was used to isolate thermophilic microbes from Rabbit Creek, Yellowstone National Park (YNP), Wyoming. Substrates for enrichment cultures including crystalline cellulose (Avicel), xylan (from Birchwood), pretreated switchgrass and Populus were used to cultivate organisms that may be of interest to lignocellulosic biofuel production.

  9. Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes. (United States)

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


    Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different conditions: 1) on a collagen coating, 2) embedded in a collagen gel, or 3) embedded in a gel put on a porous well insert. The effect of conditioned medium (CM), insulin, transferrin, selenium, and the oxygen content was assessed. Viability was tested by the metabolic dimethylthiazol-diphenyl-tetrazolium bromide assay, by flowcytometry, by phase contrast microscopy, and by transmission electron microscopy. Incubation at 21 degrees C for 75 min gave an optimal yield of 3 x 10(6) (2.0-3.8 x 10(6)) viable epithelial cells in intact crypts per seven biopsies. Embedding of crypts in a collagen gel put on a porous membrane was superior to the other methods applied [P coat-cultivated cells. CM had similar viability supporting effects to FCS. Other supplements had no effects. A simple method is presented, which makes cultivation of colonocytes obtained at endoscopy possible for up to 72 h.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yabana, Shuichi; Matuda, Akihiro [Central Research Inst. of Electric Power Industry, Abiko, Chiba (Japan). Abiko Research Lab


    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)

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

    Energy Technology Data Exchange (ETDEWEB)

    Yabana, Shuichi; Matsuda, Akihiro [Central Research Inst. of Electric Power Industry, Tokyo (Japan)


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


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


    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...

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

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


    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.

  14. Influence of different isolation methods on the survival of proximal ART restorations in primary molars after two years

    NARCIS (Netherlands)

    A.M. Kemoli; W.E. van Amerongen; G.N. Opinya


    AIM: This was to evaluate the influence of two methods of tooth-isolation on the survival rate of proximal ART restorations in the primary molars. METHODS: The study was conducted in two rural divisions in Kenya, with 7 operators randomly paired to a group of 8 assistants. A total of 804 children ea

  15. 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.


    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...

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

    Directory of Open Access Journals (Sweden)

    Tamás Baranyai

    Full Text Available 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.Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC and size exclusion chromatography (SEC.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.Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

  17. Identification of Leishmania isolates from Varzaneh city, Isfahan province, Iran using nested polymerase chain reaction method

    Directory of Open Access Journals (Sweden)

    Reza Arjmand


    Full Text Available Background: Leishmaniasis, a parasitic disease, is caused by the Leishmania genus, a protozoan parasite transmitted by sand fly arthropods. Cutaneous leishmaniasis (CL in old world is usually caused by L. major, L. tropica, and L. aethiopica complexes. One of the most important hyper endemic areas of CL in Iran is Isfahan province. Varzaneh is a city in the eastern part of Isfahan province. Due to different biological patterns of parasite strains which are distributed in the region, this study was design to identify Leishmania species from human victims using Kinetoplastid DNA as templates in a molecular PCR method. Materials and Methods: Among 186 suspected cases, 50 cases were confirmed positive by direct microscopy after Giemsa staining. Species characterization of the isolates was done using Nested- PCR as a very effective and sensitive tool to reproduce mini circle strands. Results: After Nested-PCR from all 50 cases, 560 bp bands were produced which according to products of reference strains indicate that the infection etiologic agent has been L. major. 22 (44% of patients were females and 28 (56% of them were males. Their age ranges were between 7 months and 60 years. Conclusion: According to the results of the study and the particular pattern of infection prevalent in the region, genetic studies and identification of Leishmania parasites are very important in the disease control and improvement of regional strategy of therapy protocols.

  18. Effects of phytase-assisted processing method on physicochemical and functional properties of soy protein isolate. (United States)

    Wang, Hongjian; Chen, Yeming; Hua, Yufei; Kong, Xiangzhen; Zhang, Caimeng


    Phytate is an important antinutritional factor in food products. In this study, a phytase-assisted processing method was used to produce low-phytate soybean protein isolate (SPI) samples, and their physicochemical and functional properties were examined. Hydrolysis condition at low temperature (room temperature) and pH 5.0 was better than that recommended by manufacturer (pH 5.0, 55 °C) at keeping the properties of SPI, so the former condition was selected to prepare SPI samples with phytate contents of 19.86-0.11 mg/g by prolonging hydrolysis time (0 (traditional method), 5, 10, 20, 40, and 60 min). Ash content (R(2) = 0.940), solubility (R(2) = 0.983), ζ-potential value (R(2) = 0.793), denaturation temperatures (β-conglycinin, R(2) = 0.941; glycinin, R(2) = 0.977), emulsifying activity index (R(2) = 0.983), foaming capacity (R(2) = 0.955), and trypsin inhibitor activity (R(2) = 0.821) of SPI were positively correlated with phytate content, whereas protein content (R(2) = 0.876), protein recovery (R(2) = 0.781), emulsifying stability index (R(2) = 0.953), foaming stability (R(2) = 0.919), gel hardness (R(2) = 0.893), and in vitro digestibility (R(2) = 0.969) were negatively correlated with phytate content. Simulated gastrointestinal digestion and subsequent dialysis showed that percentages of dialyzable Zn and Ca were increased with decreasing phytate content, whereas the amounts of dialyzable Zn and Ca revealed different behaviors: the former was increased and the latter was decreased. Circular dichroism spectra showed that secondary structure of SPI was changed by phytase. Compared with traditional processing method, the phytase-assisted processing method could produce SPI with lower phytate and higher protein contents, which had better in vitro digestibility and could be used to prepare gels with higher hardness by partially losing some other functional properties.

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

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


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

  20. Method for the isolation of citric acid and malic acid in Japanese apricot liqueur for carbon stable isotope analysis. (United States)

    Akamatsu, Fumikazu; Hashiguchi, Tomokazu; Hisatsune, Yuri; Oe, Takaaki; Kawao, Takafumi; Fujii, Tsutomu


    A method for detecting the undeclared addition of acidic ingredients is required to control the authenticity of Japanese apricot liqueur. We developed an analytical procedure that minimizes carbon isotope discrimination for measurement of the δ(13)C values of citric and malic acid isolated from Japanese apricot liqueur. Our results demonstrated that freeze-drying is preferable to nitrogen spray-drying, because it does not significantly affect the δ(13)C values of citric acid and results in smaller isotope discrimination for malic acid. Both 0.1% formic acid and 0.2% phosphoric acid are acceptable HPLC mobile phases for the isolation of citric and malic acid, although the δ(13)C values of malic acid exhibited relatively large variation compared with citric acid following isolation using either mobile phase. The developed procedure allows precise δ(13)C measurements of citric and malic acid isolated from Japanese apricot liqueur.

  1. Arcobacter: comparison of isolation methods, diversity, and potential pathogenic factors in commercially retailed chicken breast meat from Costa Rica. (United States)

    Fallas-Padilla, Karolina L; Rodríguez-Rodríguez, Carlos E; Fernández Jaramillo, Heriberto; Arias Echandi, María Laura


    Arcobacter species have been recognized as potential food- and waterborne pathogens. The lack of standardized isolation methods and the relatively scarce knowledge about their prevalence and distribution as emerging pathogens are due to the limitations in their detection and identification. This study aimed to determine the presence and the identification of Arcobacter in chicken breast samples commercially retailed in San José, Costa Rica, as well as to describe the adherence and invasive potential of the strains to human cells (HEp-2). Fifty chicken breast samples were collected from retail markets in the metropolitan area of the country. Six different isolation methodologies were applied for the isolation of Arcobacter. Isolation strategies consisted of combinations of enrichments in de Boer or Houf selective broths and subsequent isolation in blood agar (directly or with a previous passive membrane filtration step) or Arcobacter selective agar. Suspicious colonies were identified with a genus-specific PCR, whereas species-level identification was achieved with a multiplex PCR. The overall isolation frequency of Arcobacter was 56%. From the isolation strategies, the combination of enrichment in Houf selective broth followed by filtration on blood agar showed the best performance, with a sensitivity of 89% and a specificity of 84%. A total of 46 isolates were confirmed as Arcobacter with the genus-specific PCR, from which 27 (59%) corresponded to Arcobacter butzleri, 9 (19%) to Arcobacter cryaerophilus, and 10 (22%) were not identified with this multiplex PCR. Regarding the potential pathogenicity, 75% of the isolates presented adherence to HEp-2 cells, while only 22% were invasive to that cell line. All invasive strains were A. butzleri or nonidentified strains. The results show the presence of potentially pathogenic Arcobacter in poultry and recognize the importance it should receive as a potential foodborne pathogen from public health authorities.

  2. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik


    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...

  3. Survey of volatile oxylipins and their biosynthetic precursors in bryophytes. (United States)

    Croisier, Emmanuel; Rempt, Martin; Pohnert, Georg


    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.

  4. Whole-Genome Sequence of Bacillus sp. SDLI1, Isolated from the Social Bee Scaptotrigona depilis. (United States)

    Paludo, Camila R; Ruzzini, Antonio C; Silva-Junior, Eduardo A; Pishchany, Gleb; Currie, Cameron R; Nascimento, Fábio S; Kolter, Roberto G; Clardy, Jon; Pupo, Mônica T


    We announce the complete genome sequence ofBacillussp. strain SDLI1, isolated from larval gut of the stingless beeScaptotrigona depilis The 4.13-Mb circular chromosome harbors biosynthetic gene clusters for the production of antimicrobial compounds.

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


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


    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...

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

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    Vesna Zechner-Krpan


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


    Directory of Open Access Journals (Sweden)

    Francesco Marotta


    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.

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

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    Swathi SundarRaj


    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.

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

    DEFF Research Database (Denmark)

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


    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......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...... 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...

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

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    Noor Adila, A. K.


    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.

  11. [Physiological comportment and in vivo sensitivity of Sporothrix schenckii isolates maintained for 18 years by two preservation methods]. (United States)

    Mendoza, Mireya; Alvarado, Primavera; Díaz de Torres, Elvia; Lucena, Lilianyel; de Albornoz, María C


    We compared two methods for the preservation of fungi, Castellani's method and repeated passage in Sabouraud medium-agar, on five isolates of Sporothrix schenckii that were preserved for 18 years at room temperature by both procedures. They were evaluated for viability of the strains, growth rate, morphological and physiological characteristics, and in vitro sensitivity to iodide, itraconazole, terbinafine and posaconazole. 100% viability was observed in all of the isolates, with slower growth rate on strains preserved in water compared to strains periodically re-cultured. The typical morphological feature of these fungi was preserved by both methodologies. With regard to enzymatic activity, both groups gave urease reactions and were beta glucosidase-positive. Nevertheless, complete inhibition of the capacity to hydrolyse starch was observed only on the isolates preserved in water. This group also was more sensitive to potassium iodide at a concentration of 10 microM in the in vitro sensitivity tests.

  12. Marine Actinobacteria from the Gulf of California: diversity, abundance and secondary metabolite biosynthetic potential (United States)

    Becerril-Espinosa, Amayaly; Freel, Kelle C.; Jensen, Paul R.


    The Gulf of California is a coastal marine ecosystem characterized as having abundant biological resources and a high level of endemism. In this work we report the isolation and characterization of Actinobacteria from different sites in the western Gulf of California. We collected 126 sediment samples and isolated on average 3.1–38.3 Actinobacterial strains from each sample. Phylogenetic analysis of 136 strains identified them as members of the genera Actinomadura, Micromonospora, Nocardiopsis, Nonomuraea, Saccharomonospora, Salinispora, Streptomyces and Verrucosispora. These strains were grouped into 26–56 operational taxonomic units (OTUs) based on 16S rRNA gene sequence identities of 98–100 %. At 98 % sequence identity, three OTUs appear to represent new taxa while nine (35 %) have only been reported from marine environments. Sixty-three strains required seawater for growth. These fell into two OTUs at the 98 % identity level and include one that failed to produce aerial hyphae and was only distantly related (≤95.5 % 16S identity) to any previously cultured Streptomyces sp. Phylogenetic analyses of ketosynthase domains associated with polyketide synthase genes revealed sequences that ranged from 55 to 99 % nucleotide identity to experimentally characterized biosynthetic pathways suggesting that some may be associated with the production of new secondary metabolites. These results indicate that marine sediments from the Gulf of California harbor diverse Actinobacterial taxa with the potential to produce new secondary metabolites. PMID:23229438

  13. Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR

    NARCIS (Netherlands)

    Vlieger, de J.S.B.; Kolkman, A.J.; Ampt, K.A.M.; Commandeur, J.N.M.; Vermeulen, N.P.E.; Kool, J.; Wijmenga, S.S.; Niessen, W.M.A.; Irth, H.; Honing, M.


    This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatogra

  14. Effect of photoperiod on gibberellin biosynthetic enzymes in spinach

    Energy Technology Data Exchange (ETDEWEB)

    Gilmour, S.J.; Bleecker, A.B.; Zeevaart, J.A.D.


    The photoperiodic control of stem elongation in spinach, a long day (LD) rosette plant, is mediated by gibberellins (GAs). The early 13-hydroxylated GA biosynthetic pathway from GA/sub 12/ to GA/sub 20/ operates in spinach: GA/sub 12/ ..-->.. GA/sub 53/ ..-->.. GA/sub 44/ ..-->.. GA/sub 19/ ..-->.. GA/sub 20/. Two enzymes of this pathway, those converting GA/sub 53/ to GA/sub 44/ (GA/sub 53/ oxidase) and GA/sub 19/ to GA/sub 20/ (GA/sub 19/ oxidase), are regulated by light. The enzyme converting GA/sub 44/ to GA/sub 19/ (GA/sub 44/ oxidase) is not light-regulated. In the light GA/sub 53/ and GA/sub 18/ oxidase activities are increased, therefore causing the GA biosynthetic pathway to be turned on. This leads to the production of an active GA in LD, which causes an increase in stem elongation. Two the enzymes, GA/sub 44/ and GA/sub 53/ oxidases, can be separated from one another by anion exchange HPLC. Estimates of the molecular weights of these two enzymes based on gel filtration HPLC will be reported.

  15. The biosynthetic pathway of vitamin C in higher plants. (United States)

    Wheeler, G L; Jones, M A; Smirnoff, N


    Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.

  16. 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.


    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.

  17. Single-cell analysis and isolation for microbiology and biotechnology: methods and applications. (United States)

    Ishii, Satoshi; Tago, Kanako; Senoo, Keishi


    Various single-cell isolation techniques, including dilution, micromanipulation, flow cytometry, microfluidics, and compartmentalization, have been developed. These techniques can be used to cultivate previously uncultured microbes, to assess and monitor cell physiology and function, and to screen for novel microbiological products. Various other techniques, such as viable staining, in situ hybridization, and those using autofluorescence proteins, are frequently combined with these single-cell isolation techniques depending on the purpose of the study. In this review article, we summarize currently available single-cell isolation techniques and their applications, when used in combination with other techniques, in microbiological and biotechnological studies.

  18. Biosynthetic labeling and two-color imaging of phospholipids in cells. (United States)

    Jao, Cindy Y; Roth, Mary; Welti, Ruth; Salic, Adrian


    Phospholipids with a choline head group are abundant components of all biological membranes, performing critical functions in cellular structure, metabolism, and signaling. In spite of their importance, our ability to visualize choline phospholipids in vivo remains very limited. We present a simple and robust chemical strategy to image choline phospholipids, based on the metabolic incorporation of azidocholine analogues, that accurately reflects the normal biosynthetic incorporation of choline into cellular phospholipids. Azidocholine-labeled phospholipids can be imaged in cells with high sensitivity and resolution, following derivatization with fluorophores, by bio-orthogonal chemical reactions compatible with live-cell imaging. We used this method to visualize the subcellular localization of choline phospholipids. We also demonstrate that double metabolic labeling with azidocholine and propargylcholine allows sensitive two-color imaging of choline phospholipids. Our method represents a powerful approach to directly image phospholipids, and to study their dynamics in cells and tissues.

  19. Isolated yeast promoter sequence and a method of regulated heterologous expression (United States)

    Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.


    The present invention provides the promoter clone discovery of a glucoamylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated glucoamylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

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

    NARCIS (Netherlands)

    P.J.M. Valk (Peter); M. Joosten (Marieke); Y. Vankan; H.R. Delwel (Ruud); B. Löwenberg (Bob)


    textabstractProto-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. W

  1. Identification of Malassezia Species Isolated from Patients with Pityriasis Versicolor Using PCR-RFLP Method in Markazi Province, Central Iran.

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    Mojtaba Didehdar


    Full Text Available The lipophilic yeasts of Malassezia species are members of the normal skin microbial that are cause of pityriasis versicolor. Pityriasis versicolor is a common superficial fungal infection with world-wide distribution. The phenotypic methods for identification of Malassezia species usually are time consuming and unreliable to differentiate newly identified species. But DNA-based techniques rapidly and accurately identified Malassezia species. The purpose of this study was isolation and identification of Malassezia Species from patients with pityriasis versicolor by molecular methods in Markazi Province, Central Iran in 2012.Mycologic examinations including direct microscopy and culture were performed on clinical samples. DNA extraction was performed from colonies. The ITS1 region of rDNA from isolates of Malassezia species were amplified by PCR reaction. The PCR were digested by Cfo I enzyme.From 70 skin samples, were microscopically positive for Malassezia elements, 60 samples were grown on culture medium (85.7%. Using PCR-RFLP method, that was performed on 60 isolates, 37(61.6% M. globosa, 14(23.3% M. furfur, 5(8.4% M. sympodialis and 4(6.7% M. restrictawere identified. In one case was isolated M. globosa along with M. restricta.The PCR-RFLP method is a useful and reliable technique for identification of differentiation of Malas-sezia species.

  2. Performance of Five Phenotypical Methods for Identification of Candida Isolates from Clinical Materials

    Directory of Open Access Journals (Sweden)

    F Zaini


    Full Text Available Although Candida albicans is the most common etiologic agent of candidiasis, C. dubliniensis, has been emerged, as another pathogen resembles C. albicans in many phenotypic aspects and noted for its in vitro potential for fluconazole resistance. Since there was no evidence of any report about detection of this organism in Iran, this study was designed to use of five different tests for identification of Candida species with special reference to C. dubliniensis among 313 suspected Candida isolates in Tehran, capital of Iran. Overall, 199 (63.6% C. albicans and 114 (36.6% Candida spp. were identified. All 199 C. albicans isolates were found germ tube and chlamydospore positive. Different shades of green color colonies were yielded on CHROMagar Candida of which 23 (11.6% showed dark green color indicative of C. dubliniensis. All but four C. albicans isolates grew well at 45 °C. These 4 isolates beyond to 23 dark green colony producers were suspected of being C.dubliniensis, later examined by API 20C AUX system. The results indicated that all 27 isolates were able to assimilate both xylose and α-methyl-D-glucoside, therefore these isolates were identified as C. albicans. Overall, C. dubliniensis had not been found in present study. It must be concluded that no single phenotypic test has proven to be highly effective, and the use of several tests may be necessary of these two closely related Candida species for definitive identification.

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

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


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

  4. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. (United States)

    Greening, David W; Xu, Rong; Ji, Hong; Tauro, Bow J; Simpson, Richard J


    Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).

  5. Typing of Campylobacter jejuni Isolated from Turkey by Genotypic Methods, Antimicrobial Susceptibility, and Virulence Gene Patterns: A Retrospective Study. (United States)

    Manfreda, Gerardo; Parisi, Antonio; De Cesare, Alessandra; Mion, Domenico; Piva, Silvia; Zanoni, Renato G


    In this retrospective study, typing ability, discriminatory power, and concordance between typing results obtained on 123 Campylobacter jejuni turkey isolates, collected in 1998, within 14 different farms, applying multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), antibiotic resistance profile, and virulence gene pattern, were assessed and compared. Overall, 33 sequence types, 28 pulsotypes, 10 resistotypes, and 5 pathotypes were identified. MLST and PFGE showed the better discriminatory ability (i.e., Simpson's diversity index >0.90) as well as unidirectional (i.e., Wallace and adjusted Wallace coefficients >0.86) and bidirectional (i.e., adjusted Rand coefficient >0.60) concordance. Moreover, both methods showed a good unidirectional and bidirectional concordance with the resistotype. On the contrary, the congruence of both genotyping methods and resistotype with the pathotype seemed due to chance alone. A clonal relationship was identified among 66.7% of the isolates. Furthermore, 59.7% of the investigated isolates were resistant to two or more antimicrobials and 92% to tetracycline. All the isolates harbored cadF and pldA genes, whereas a flaA gene product and a cdtB gene product were amplified from 85.4% and 79.7% of the isolates, respectively, using the primers designed by Bang et al. (2003). The results of this study clarify the level of genetic diversity among the C. jejuni originating from turkeys. MLST level of correlation with PFGE, resistotype, and pathotype is assessed. This result supports the selection of type and number of typing methods to use in epidemiological studies. Finally, the identification of clonal complexes (i.e., groups of profiles differing by no more than one gene from at least one other profile of the group using the entire Campylobacter MLST database) shared between turkey and human isolates suggests that turkeys could be a possible source of Campylobacter infection.

  6. Comparison of genotypic and phenotypic characterization methods for Pasteurella multocida isolates from fatal cases of bovine respiratory disease. (United States)

    Taylor, Jared D; Fulton, Robert W; Dabo, S Mady; Lehenbauer, Terry W; Confer, Anthony W


    Bovine respiratory disease (BRD) is the most costly disease of beef cattle in North America. Because Pasteurella multocida is a commensal of the upper respiratory tract, it is generally considered an opportunistic pathogen. However, studies in swine indicated that there may be a limited number of strains associated with disease, suggesting that some are more virulent than others. Although this may also be true of isolates from cattle, appropriate typing methods must be established before this possibility can be investigated. The purpose of this study was to compare effectiveness of polymerase chain reaction (PCR) fingerprinting to more traditional approaches for typing bovine P. multocida isolates. Isolates were obtained from 41 cases of fatal BRD and subjected to random amplified polymorphic DNA PCR (RAPD-PCR), whole cell protein (WCP) profiles, outer membrane protein (OMP) profiles, and serotyping. The discrimination index was calculated for each typing method and combinations of each using Simpson's index of diversity. Correlation coefficients were calculated to assess concordance between classification results achieved through genotypic (RAPD-PCR) and phenotypic (WCP, OMP, and serotyping) approaches. All characterization methods were capable of discriminating between isolates. However, there was poor concordance between techniques. There were also few significant associations between typing results and epidemiologic data. Random amplified polymorphic DNA PCR was validated as being a repeatable and reliable means of discriminating between P. multocida isolates obtained from cattle. Isolates obtained from fatal cases of BRD in calves in a commercial feedlot demonstrated significant diversity, justifying additional investigation into whether P. multocida is a strictly opportunistic pathogen in cattle.

  7. IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes (United States)

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


    Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) ( Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery. PMID:27903896

  8. Comparison The Effects of Two Monocyte Isolation Methods,Plastic Adherence and Magnetic Activated Cell Sorting Methods,on Phagocytic Activity of Generated Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Behnaz Asadi


    Full Text Available Objective: It is believed that monocyte isolation methods and maturation factors affect the phenotypic and functional characteristics of resultant dendritic cells (DC. In the present study, we compared two monocyte isolation methods, including plastic adherence-dendritic cells (Adh-DC and magnetic activated cell sorting- dendritic cells (MACS-DC, and their effects on phagocytic activity of differentiated immature DCs (immDCs.Materials and Methods: In this experimental study, immDCs were generated from plastic adherence and MACS isolated monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin 4 (IL-4 in five days. The phagocytic activity of immDCs was analyzed by fluorescein isothiocyanate (FITC-conjugated latex bead using flow cytometry. One way ANOVA test was used for statistical analysis of differences among experimental groups, including Adh-DC and MACS-DC groups.Results: We found that phagocytic activity of Adh-DC was higher than MACS-DC, whereas the mean fluorescence intensity (MFI of phagocytic cells was higher in MACS-DC (p<0.05.Conclusion: We concluded that it would be important to consider phagocytosis parameters of generated DCs before making any decision about monocyte isolation methods to have fully functional DCs.

  9. Alanylclavam Biosynthetic Genes Are Clustered Together with One Group of Clavulanic Acid Biosynthetic Genes in Streptomyces clavuligerus▿ § (United States)

    Zelyas, Nathan J.; Cai, Hui; Kwong, Thomas; Jensen, Susan E.


    Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway. PMID:18931110

  10. Isolated Fungal Promoters and Gene Transcription Terminators and Methods of Protein and Chemical Production in a Fungus (United States)

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


    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.

  11. 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


    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.

  12. Comparison of Paraffin Bait, Humic Acid Vitamin B Agar and Paraffin Agar Methods to Isolate Nocardia from Soil

    Directory of Open Access Journals (Sweden)

    Rasoulinasab, M. (MSc


    Full Text Available Background and Objective: The Isolation of Nocardia species is complex and time-consuming, which is due to rapid growth of adjacent bacteria. Because of the importance of a specific medium with the ability of controlling intrusive microorganisms, this study aimed at comparing three laboratory methods to introduce the reliable isolation technique for Nocardia species. Material and Methods: The soil samples were collected from different regions of Tehran province, Iran, and carefully transferred to the laboratory. The samples were cultured in three different media including Paraffin Baiting,Humic acid vitamin B agar and Paraffin agar, and incubated for 3-4 weeks at 35 °C. Results: Of 110 soil samples, 31 Nocardia isolates (28.18% were obtained from the media including Paraffin Baiting, (19; 17.27%, Humic acid and vitamin B agar (4; 3.63%, and Paraffin agar, (8; 7.27%. Conclusion: because of high rate of isolation, low cost and the clearance of colonies suspected nocardia, Paraffin Bait technique is more reliable and efficient compared to the other methods. Key words: Nocardia; Soil; Paraffin Baiting; Humic Acid Vitamin B

  13. 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


    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.

  14. 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.


    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

  15. Detection of additional genes of the patulin biosynthetic pathway in Penicillium griseofulvum (United States)

    Genes in the patulin biosynthetic pathway are likely to be arranged in a cluster as has been found for biosynthetic pathways of other mycotoxins. The mycotoxin patulin, common in apples and apple juice, is most often associated with Penicillium expansum. However, of 15 fungal species capable of sy...

  16. Combination cellulose plate (non-agar solid support) and agar plate method improves isolation of fungi from soil. (United States)

    Nonaka, Kenichi; Todaka, Nemuri; Ōmura, Satoshi; Masuma, Rokuro


    This is the first report describing the improved isolation of common filamentous fungi via a method combining cellulose plate and agar plate system. A cellulose plate is a porous plate made of nanofibrous crystaline cellulose. Isolating fungi from soils using these types of media separately resulted in the number of fungal colonies appearing on cellulose plates being lower than that on agar plates. However, the number of actual fungal species isolated using cellulose plates alone was more or less the same as that found using agar plates. Significantly, the diversity of isolates using a combination of the two media was greater than using each media individually. As a result, numerous new or rare fungal species with potential, including previously proposed new species, were isolated successfully in this way. All fungal colonies, including the Penicillium species, that appeared on the cellulose plate penetrated in potato dextrose were either white or yellow. Cultivation on cellulose plates with added copper ion overcomes the change in coloration, the colonies appearing as they do following cultivation on potato dextrose agar.

  17. A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model. (United States)

    Gesteira, Tarsis F; Coulson-Thomas, Vivien Jane; Ogata, Fernando T; Farias, Eduardo H C; Cavalheiro, Renan P; de Lima, Marcelo A; Cunha, Gabriel L A; Nakayasu, Ernesto S; Almeida, Igor C; Toma, Leny; Nader, Helena B


    Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.

  18. 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


    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

  19. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites. (United States)

    Komatsu, Mamoru; Komatsu, Kyoko; Koiwai, Hanae; Yamada, Yuuki; Kozone, Ikuko; Izumikawa, Miho; Hashimoto, Junko; Takagi, Motoki; Omura, Satoshi; Shin-ya, Kazuo; Cane, David E; Ikeda, Haruo


    An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

  20. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods. (United States)

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A


    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  1. Application of CPC and related methods for the isolation of natural substances--a review. (United States)

    Kedzierski, Bartosz; Kukuła-Koch, Wirginia; Głowniak, Kazimierz


    A review of research on the isolation of various alkaloids from plant material by centrifugal partition chromatography (CPC) and related preparative techniques was made, in order to provide various conditions for separation of these important plant derived secondary metabolites. First of all, the construction of the CPC apparatus was presented as well as the principle of isolation of natural products with its help, and then the influence of operating apparatus parameters on the separation efficiency. Finally, a review of the alkaloids separation conditions was made, specifying used parameters and best solvent system.

  2. Limiting Cholesterol Biosynthetic Flux Spontaneously Engages Type I IFN Signaling. (United States)

    York, Autumn G; Williams, Kevin J; Argus, Joseph P; Zhou, Quan D; Brar, Gurpreet; Vergnes, Laurent; Gray, Elizabeth E; Zhen, Anjie; Wu, Nicholas C; Yamada, Douglas H; Cunningham, Cameron R; Tarling, Elizabeth J; Wilks, Moses Q; Casero, David; Gray, David H; Yu, Amy K; Wang, Eric S; Brooks, David G; Sun, Ren; Kitchen, Scott G; Wu, Ting-Ting; Reue, Karen; Stetson, Daniel B; Bensinger, Steven J


    Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

  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 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...... of chloroplast enzymes can play a role not only in enzyme activity and redox sensitivity but also in protein folding and stability upon oxidation. Several redox sensitive enzymes identified in this study can serve as potential targets to control the carbon flux to and from starch during the day and night...

  4. Functional Analysis of the Fusarielin Biosynthetic Gene Cluster

    Directory of Open Access Journals (Sweden)

    Aida Droce


    Full Text Available Fusarielins are polyketides with a decalin core produced by various species of Aspergillus and Fusarium. Although the responsible gene cluster has been identified, the biosynthetic pathway remains to be elucidated. In the present study, members of the gene cluster were deleted individually in a Fusarium graminearum strain overexpressing the local transcription factor. The results suggest that a trans-acting enoyl reductase (FSL5 assists the polyketide synthase FSL1 in biosynthesis of a polyketide product, which is released by hydrolysis by a trans-acting thioesterase (FSL2. Deletion of the epimerase (FSL3 resulted in accumulation of an unstable compound, which could be the released product. A novel compound, named prefusarielin, accumulated in the deletion mutant of the cytochrome P450 monooxygenase FSL4. Unlike the known fusarielins from Fusarium, this compound does not contain oxygenized decalin rings, suggesting that FSL4 is responsible for the oxygenation.

  5. Extending the biosynthetic repertoires of cyanobacteria and chloroplasts

    DEFF Research Database (Denmark)

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


    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...

  6. An efficient screening method for the isolation of heterotrophic bacteria influencing growth of diatoms under photoautotrophic conditions. (United States)

    Zecher, Karsten; Jagmann, Nina; Seemann, Philipp; Philipp, Bodo


    Interactions between photoautotrophic diatoms and heterotrophic bacteria are important for the biogeochemical C-cycle in the oceans. Additionally, biofilms formed by diatoms and bacteria are the initiating step of biofouling processes, which causes high costs in shipping. Despite this ecological and economical importance, the knowledge about biochemical and molecular mechanisms underlying these interkingdom interactions is relatively small. For analyzing these mechanisms, laboratory model systems are required. In this study, an efficient screening method for isolating bacteria influencing photoautotrophic diatom growth was established. First, diatom cultures of Phaeodactylum tricornutum and Thalassiosira pseudonana were made axenic by applying β-lactam antibiotics. Second, a non-invasive method for measuring growth of multiple parallel diatom cultures by chlorophyll fluorescence was established. This method allowed semi-quantitative chlorophyll determination of cultures with up to 3 μg (chlorophyll) ml(-1). Axenic diatom cultures were then used for enriching bacteria and led to the isolation of 24 strains influencing growth of both diatom strains in various ways. For example, Rheinheimera sp. strain Tn16 inhibited growth of T. pseudonana, while it stimulated growth and cell aggregation of P. tricornutum. Thus, this screening method is appropriate for isolating heterotrophic bacteria showing different interactions with different diatom species ranging from synergistic to antagonistic. In consecutive applications, this method will be useful to screen for bacterial mutants with altered phenotypes regarding the influence on diatom growth.

  7. 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.

  8. Resorbable biosynthetic mesh for crural reinforcement during hiatal hernia repair. (United States)

    Alicuben, Evan T; Worrell, Stephanie G; DeMeester, Steven R


    The use of mesh to reinforce crural closure during hiatal hernia repair is controversial. Although some studies suggest that using synthetic mesh can reduce recurrence, synthetic mesh can erode into the esophagus and in our opinion should be avoided. Studies with absorbable or biologic mesh have not proven to be of benefit for recurrence. The aim of this study was to evaluate the outcome of hiatal hernia repair with modern resorbable biosynthetic mesh in combination with adjunct tension reduction techniques. We retrospectively analyzed all patients who had crural reinforcement during repair of a sliding or paraesophageal hiatal hernia with Gore BioA resorbable mesh. Objective follow-up was by videoesophagram and/or esophagogastroduodenoscopy. There were 114 patients. The majority of operations (72%) were laparoscopic primary repairs with all patients receiving a fundoplication. The crura were closed primarily in all patients and reinforced with a BioA mesh patch. Excessive tension prompted a crural relaxing incision in four per cent and a Collis gastroplasty in 39 per cent of patients. Perioperative morbidity was minor and unrelated to the mesh. Median objective follow-up was one year, but 18 patients have objective follow-up at two or more years. A recurrent hernia was found in one patient (0.9%) three years after repair. The use of crural relaxing incisions and Collis gastroplasty in combination with crural reinforcement with resorbable biosynthetic mesh is associated with a low early hernia recurrence rate and no mesh-related complications. Long-term follow-up will define the role of these techniques for hiatal hernia repair.

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

    Directory of Open Access Journals (Sweden)

    JungReem Woo


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

  10. A New Method for the Isolation of Ergosterol and Peroxyergosterol as Active Compounds of Hygrophoropsis aurantiaca and in Vitro Antiproliferative Activity of Isolated Ergosterol Peroxide

    Directory of Open Access Journals (Sweden)

    Renata Nowak


    Full Text Available In the present study, ergosterol peroxide and ergosterol were isolated for the first time from fresh fruit bodies of Hygrophoropsis aurantiaca (False Chanterelle. The substances were characterized mainly by spectroscopic methods (1H-NMR, 13C-NMR, DEPT-45, DEPT-90, DEPT-135, 2D-NMR. In our study, a new specific thin layer chromatographic method was developed for determination of ergosterol and ergosterol peroxide in H. aurantiaca extract. The method is based on the separation of n-hexane extract on silica gel (Silica Gel G TLC plates using the optimized solvent system toluene/ethyl acetate (3:1; v/v. The main advantages of the developed method are the simplicity of operation and the low cost. The in vitro study results revealed the antiproliferative properties of ergosterol peroxide against LS180 human colon cancer cells. The described effect was attributed both to altered mitochondrial activity and decreased DNA synthesis. Additionally, in the same concentration range the investigated compound was not toxic to CCD 841 CoTr human colon epithelial cells. The present study suggests that fruit bodies of H. aurantiaca have great potential for producing substances and extracts with potential applications in medicine.

  11. 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


    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...

  12. Development of a Low-Cost Method for Whole-Spacecraft Isolation of Small Satellites (United States)


    17 Figure 2.6 Transmissibility of NPSCuL (SDOF Model) and ABC Plate (2DOF Model...92 Figure 4.30 Transmissibility of Isolated NPSCuL (SDOF Model) and ABC Plate (2DOF Model...MPF Mode Participation Factor MS Margin of Safety NASA National Air and Space Administration NEA Non-Explosive Actuator NLAS Nanosatellite Launch

  13. Method of defining isolation parameters in 1000 Watt symmetric electricity network

    Directory of Open Access Journals (Sweden)

    Samat Amurgaliev


    Full Text Available The authors define parameters for isolation three-phase electric network to provide safety conditions for personnel working at open-operated coal deposits. The authors conclude the technique provides satisfactory accuracy of the parameters’ defining, as well as simplicity and safety of coal mining works with electric network having pressure up to 1000 Watt.

  14. Genetic engineering, high resolution mass spectrometry and nuclear magnetic resonance spectroscopy elucidate the bikaverin biosynthetic pathway in Fusarium fujikuroi. (United States)

    Arndt, Birgit; Studt, Lena; Wiemann, Philipp; Osmanov, Helena; Kleigrewe, Karin; Köhler, Jens; Krug, Isabel; Tudzynski, Bettina; Humpf, Hans-Ulrich


    Secondary metabolites of filamentous fungi can be highly bioactive, ranging from antibiotic to cancerogenic properties. In this study we were able to identify a new, yet unknown metabolite produced by Fusarium fujikuroi, an ascomycetous rice pathogen. With the help of genomic engineering and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS) followed by isolation and detailed structure elucidation, the new substance could be designated as an unknown bikaverin precursor, missing two methyl- and one hydroxy group, hence named oxo-pre-bikaverin. Though the bikaverin gene cluster has been extensively studied in the past, elucidation of the biosynthetic pathway remained elusive due to a negative feedback loop that regulates the genes within the cluster. To decipher the bikaverin biosynthetic pathway and to overcome these negative regulation circuits, the structural cluster genes BIK2 and BIK3 were overexpressed independently in the ΔΔBIK2/BIK3+OE::BIK1 mutant background by using strong constitutive promoters. Using the software tool MZmine 2, the metabolite profile of the generated mutants obtained by HPLC-HRMS was compared, revealing further intermediates.

  15. Simple and Rapid Method of Isolating Humic Acids from Tropical Peat Soils (Saprists

    Directory of Open Access Journals (Sweden)

    Shamsuddin Rosliza


    Full Text Available Problem Statement: The isolation (extraction, fractionation and purification of humic acids (HA from soils is laborious, time consuming and expensive. The extraction, fractionation and purification periods of these substances vary from 12 h-7 days. In order to facilitate production of HA at competitive cost, this study was conducted to investigate whether a simple and rapid procedure could be developed for isolation of HA from well decomposed tropical peat soils (Saprists. Approach: A 0.1 M KOH was used to isolate HA of air dry peat soil at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h extraction periods after which samples (liquid obtained after centrifugation at 16,211 G for 15 min were fractionated (using 6 M HCl at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. Samples were purified by washing them five times using distilled water instead of using HCl, HF, and an expensive process called dialysis that requires 1 to 7 days to purify HA. Each washing time was 10 min. Standard procedures were used to ascertain the purity (Ash, C, E4/E6, carboxylic, phenolic, total acidity, and K, Ca, Mg, and Na and quantity of HA yield. Statistical Analysis System (SAS was used for statistical analysis. Results: Although there was a linear relationship between extraction period and HA yield, there was no relationship between fractionation period and yield of HA. Distilled water used in this study was effective in purifying HA of the Saprists within 1 h without altering the true chemical nature of HA as it significantly reduced the mineral content of HA. Besides, C, E4/E6, carboxylic, phenolic, and total acidity of the isolated HA were typical of standard ones. Conclusion: The isolation of HA from peat soils can be reduced to 9 h (4 h extraction period, 4 h fractionation period and 1 h purification period instead of the existing range of 1 to 7 days.

  16. 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.

  17. 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


    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.

  18. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis (United States)

    de Castro Melo, Poliana; Ferreira, Luciano Menezes; Filho, Antônio Nader; Zafalon, Luiz Francisco; Vicente, Hinig Isa Godoy; de Souza, Viviane


    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. PMID:24159293

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

    Directory of Open Access Journals (Sweden)

    Elijah Johnson


    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.

  20. 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.

  1. Cloning and expression of anthocyanin biosynthetic genes in red and white pomegranate. (United States)

    Zhao, Xueqing; Yuan, Zhaohe; Feng, Lijuan; Fang, Yanming


    Exterior fruit color is an important trait for the evaluation of pomegranate fruit quality, but the molecular mechanism underlying the variation in color between red- and white-fruited pomegranate is poorly understood. In this study, full-length cDNA clones encoding enzymes involved in anthocyanin biosynthesis-such as chalcone synthase, chalcone isomerase, flavanone 3-hydoxylase, dihydroflavonol 4-reductase, anthocyanidin synthase (ANS), UDP-glucose-flavonoid 3-O-glucosyltransferase, and the R2R3 MYB transcription factor PgMYB-were isolated from fruit peels. In addition, transcript levels of anthocyanin biosynthetic genes were quantitatively measured by real-time PCR in red and white fruits. In both cultivars, two expression peaks for structural genes were detected during fruit development, whereas only one peak was observed-during early development-for PgMYB. While PgMYB is important for flavonoid biosynthesis, other transcription factors appear to also be necessary for the regulation of anthocyanin biosynthesis. No anthocyanins were detected in the white cultivar. Peels of white fruits contained transcripts of all identified genes except for PgANS, suggesting that the lack of PgANS expression may be the main factor responsible for the absence of anthocyanins in white pomegranate. PgANS may be the key gene involved in anthocyanin biosynthesis in pomegranate fruit.

  2. Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds. (United States)

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


    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.

  3. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants. (United States)

    Ghosh, Raju; Paul, Sujay; Ghosh, Subrata Kumar; Roy, Anirban


    A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

  4. Improved culture methods for isolation of Salmonella organisms from swine feces

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Mortensen, Alicja


    . Procedure-4 experiments were performed to evaluate the following: 1) diagnostic sensitivity of the selective preenrichment and rapid isolation novel technology (SPRINT) protocol, compared with that of the modified ISO protocol; 2) detection limit of the SPRINT protocol for Salmonella organisms; 3) use...... of preenrichment of samples between the use of UPE broth or BPW. Conclusions and Clinical Relevance-The SPRINT protocol may provide a faster alternative for isolation of Salmonella organisms from swine fecal samples. Furthermore, the use of TTN broth instead of SC broth may increase the sensitivity of the modified......Objective-To compare 3 alternative culture techniques for the detection of Salmonella organisms in swine feces with a modification of the international Standard Organization (ISO) 6579 standard protocol. Sample Population-Fecal samples from swine herds suspected of having Salmonella infections...

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



    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 yi...

  6. Degradation pathways of low-ethoxylated nonylphenols by isolated bacteria using an improved method. (United States)

    Zhang, Yu; Gu, Xin; Zhang, Jing; Yang, Min


    Nonylphenol ethoxylates (NPEOs) with low ethoxylation degree (NPav₂EO; containing two ethoxy units on average) and estrogenic properties are the intermediate products of nonionic surfactant NPEOs. To better understand the environmental fate of low-ethoxylated NPEOs, phylogenetically diverse low-ethoxylated NPEO-degrading bacteria were isolated from activated sludge using gellan gum as the gelling reagent. Four isolates belonging to four genera, i.e., Pseudomonas sp. NP522b in γ-Proteobacteria, Variovorax sp. NP427b and Ralstonia sp. NP47a in β-Proteobacteria, and Sphingomonas sp. NP42a in α-Proteobacteria were acquired. Ralstonia sp. NP47a or Sphingomonas sp. NP42a, have not been reported for the degradation of low-ethoxylated NPEOs previously. The biotransformation pathways of these isolates were investigated. The first three strains (NP522b, NP427b, and NP47a) exhibited high NPav₂EO oxidation ability by oxidizing the polyethoxy (EO) chain to form low-ethoxylated nonylphenoxy carboxylates, and then further oxidizing the alkyl chain to form carboxyalkylphenol polyethoxycarboxylates. Furthermore, Sphingomonas sp. NP42a degraded NPav2EO through a nonoxidative pathway with nonylphenol monoethoxylate as the dominant product.

  7. Sensitivity of isolated eggs of pond snails: a new method for toxicity assays and risk assessment. (United States)

    Liu, Tengteng; Koene, Joris M; Dong, Xiaoxiao; Fu, Rongshu


    The concentration of heavy metals in the environment is normally low. We here address whether using the development of isolated pond snail Radix auricularia eggs would provide a more sensitive endpoint and whether the gelatinous matrix of the egg mass surrounding the eggs indeed protects the snail embryos. In the present study, artificial removal of the gelatinous matrix of egg masses greatly increased the sensitivity of developing eggs to a heavy metal (cadmium). The sensitivity of isolated eggs to cadmium was determined using several convenient endpoints, including mortality, hatching rate, and heart rate, with an acute toxicity test and a subchronic test. In the acute toxicity test, a 96-h LC(50) value of 58.26 μg/L cadmium was determined. In the subchronic toxicity test, sublethal effects in terms of a significant reduction in hatching rate could be found in the 25-μg/L treatment, and a significant decrease of heart rate was observed in both treatments (5 and 25 μg/L). The high sensitivity of isolated eggs indicates that such tests can be efficient for toxicity assays and risk assessment, although one needs to keep in mind that the ecologically relevant measure of toxicity will be how eggs are affected when they are still inside the egg mass.

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

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby


    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].

  9. Isolation and characterization of primary microglia from post-natal murine brain tissues: a comparison of two methods. (United States)

    Jose, Shinsmon; Tan, Shi Wei; Tong, Chih Kong; Vidyadaran, Sharmili


    Microglia are resident macrophages of the central nervous system (CNS). Apart from playing vital roles as sentinel cells, they are crucial in physiological processes such as synaptic pruning during brain development. CNS disorders require an understanding of the contribution of each cellular compartment to the pathogenesis. Elucidating the role of microglia in disease development and progression in the intricate CNS environment is technically challenging and requires the establishment of reliable, reproducible techniques to isolate and culture microglia. A number of different protocols have been developed for isolation of neonatal microglia and here we compare two widely used methods, namely, mild trypsinization and EasySep® magnetic separation. EasySep® magnetic separation provided higher microglia yield, and flow cytometric evaluation of CD11b and F4/80 markers revealed that EasySep® separation method also produced significantly higher purity compared to mild trypsinization. Microglia isolated using EasySep® separation method were functional, as demonstrated by the generation of nitric oxide, IL-6, TNF-α, and MCP-1 in response to lipopolysaccharide stimulation. In summary, this study has revealed that magnetic separation is superior to mild trypsinization in terms of yield and purity of microglia.

  10. Electrophoretic and zymographic characterization of proteins isolated by various extraction methods from ejaculated and capacitated boar sperms. (United States)

    Zigo, Michal; Jonáková, Věra; Maňásková-Postlerová, Pavla


    The presented work focuses on electrophoretic and zymographic characterization of boar sperm proteins isolated by various extraction methods and on comparison of the protein profiles obtained from ejaculated and in vitro capacitated spermatozoa. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated with the following agents: 1% v/v Triton X-100, 1% v/v Triton X-114, 2% v/v acetic acid, 1% m/v sodium dodecyl sulphate (SDS), 30 mM N-octyl-β-D-glucopyranoside (OBG), rehydration buffer (RHB) for isoelectric focusing and finally by the freezing-thawing approach. The extracts were characterized in terms of 1-DE, 2-DE protein profiles, 1-DE glycoprotein staining and proteinase and hyaluronidase substrate zymographic profiles. The results have shown quantitative and qualitative differences in 1-DE protein and glycoprotein profiles with respect to the employed isolation approach. These differences were seen even more clearly in 2-DE protein profiles, where it was possible to distinguish the presence/absence, changes in relative abundance and pI/M(r) shifts of various protein spots. Proteinase and hyaluronidase zymograms supported the prediction that various isolation protocols result in various profiles of enzymatically active molecules.

  11. Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples (United States)

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


    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

  12. 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)


    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.

  13. Isolation and determination of absolute configurations of insect-produced methyl-branched hydrocarbons. (United States)

    Bello, Jan E; McElfresh, J Steven; Millar, Jocelyn G


    Although the effects of stereochemistry have been studied extensively for volatile insect pheromones, little is known about the effects of chirality in the nonvolatile methyl-branched hydrocarbons (MBCHs) used by many insects as contact pheromones. MBCHs generally contain one or more chiral centers and so two or more stereoisomeric forms are possible for each structure. However, it is not known whether insects biosynthesize these molecules in high stereoisomeric purity, nor is it known whether insects can distinguish the different stereoisomeric forms of MBCHs. This knowledge gap is due in part to the lack of methods for isolating individual MBCHs from the complex cuticular hydrocarbon (CHC) blends of insects, as well as the difficulty in determining the absolute configurations of the isolated MBCHs. To address these deficiencies, we report a straightforward method for the isolation of individual cuticular hydrocarbons from the complex CHC blend. The method was used to isolate 36 pure MBCHs from 20 species in nine insect orders. The absolute stereochemistries of the purified MBCHs then were determined by digital polarimetry. The absolute configurations of all of the isolated MBCHs were determined to be (R) by comparison with a library of synthesized, enantiomerically pure standards, suggesting that the biosynthetic pathways used to construct MBCHs are highly conserved within the Insecta. The development of a straightforward method for isolation of specific CHCs will enable determination of their functional roles by providing pure compounds for bioassays.

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

    Directory of Open Access Journals (Sweden)

    Kenneth W. Witwer


    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.


    Directory of Open Access Journals (Sweden)

    Santosh Patil


    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

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

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


    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.

  17. Exploitation of the Streptomyces coelicolor A3(2) genome sequence for discovery of new natural products and biosynthetic pathways. (United States)

    Challis, Gregory L


    Streptomyces, and related genera of Actinobacteria, are renowned for their ability to produce antibiotics and other bioactive natural products with a wide range of applications in medicine and agriculture. Streptomyces coelicolor A3(2) is a model organism that has been used for more than five decades to study the genetic and biochemical basis for the production of bioactive metabolites. In 2002, the complete genome sequence of S. coelicolor was published. This greatly accelerated progress in understanding the biosynthesis of metabolites known or suspected to be produced by S. coelicolor and revealed that streptomycetes have far greater potential to produce bioactive natural products than suggested by classical bioassay-guided isolation studies. In this article, efforts to exploit the S. coelicolor genome sequence for the discovery of novel natural products and biosynthetic pathways are summarized.

  18. Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer Streptomyces collinus Tü 365

    DEFF Research Database (Denmark)

    Iftime, Dumitrita; Kulik, Andreas; Härtner, Thomas;


    Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein...... metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized...... biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary...

  19. An alternative method to achieve one-lung ventilation by surgical pneumothorax in difficult lung isolation patient: a case report. (United States)

    Yeh, Pin-Hung; Hsu, Po-Kai


    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.

  20. A Reproducible Method for Isolation and In Vitro Culture of Functional Human Lymphoid Stromal Cells from Tonsils (United States)

    Bar-Ephraim, Yotam E.; Konijn, Tanja; Gönültas, Mehmet; Mebius, Reina E.


    The stromal compartment of secondary lymphoid organs is classicaly known for providing a mechanical scaffold for the complex interactions between hematopoietic cells during immune activation as well as for providing a niche which is favorable for survival of lymphocytes. In recent years, it became increasingly clear that these cells also play an active role during such a response. Currently, knowledge of the interactions between human lymphoid stroma and hematopoietic cells is still lacking and most insight is based on murine systems. Although methods to isolate stromal cells from tonsils have been reported, data on stability in culture, characterization, and functional properties are lacking. Here, we describe a reproducible and easy method for isolation and in vitro culture of functional human lymphoid stromal cells from palatine tonsils. The cells isolated express markers and characteristics of T cell zone fibroblastic reticular cells (FRCs) and react to inflammatory stimuli by upregulating inflammatory cytokines and chemokines as well as adhesion molecules, as previously described for mouse lymphoid stroma. Also, cultured tonsil stromal cells support survival of human innate lymphoid cells, showing that these stromal cells can function as bone fide FRCs, providing a favorable microenvironment for hematopoietic cells. PMID:27907202

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

    Directory of Open Access Journals (Sweden)

    Yunlong eLu


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

  2. 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


    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.)


    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová


    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.

  4. A method for the isolation of protoplasts from grape berry mesocarp tissue. (United States)

    Fontes, Natacha; Delrot, Serge; Gerós, Hernâni


    As single cell systems, protoplasts have been used in physiological, biochemical and molecular studies aiming towards the investigation, improvement or modification of plants. In grapevine, protoplasts have been isolated from several source tissues but not from grape berry, a major challenge given the uniqueness of grape fruit for human diet and wine production. Also, as the ripe grape berry has long been considered a 'small bag of sugary water' without cell compartmentation and/or membrane integrity, the isolation of intact cells from the mesocarp is of special scientific significance. Protoplasting from grape berry mesocarp cells was achieved with cellulase and pectolyase digestion, followed by differential and gradient centrifugations; however, given the special characteristics of berry tissue, cell wall digestion and protoplast purification were performed in a special environment to maintain their integrity and viability. Light and epifluorescence microscopy revealed the spatial organization of the cytoplasm, where an intricate acidic vacuolar apparatus predominates supporting the idea that berry softening during ripening is not strictly associated with loss in compartmentation and/or membrane integrity. Following the worldwide economical and social importance of wine in modern days, grape berry protoplasts are a major advance for both basic research of fruit ripening and biotechnological applications.

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

    Directory of Open Access Journals (Sweden)

    Dodoš Tanja


    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

  6. Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8

    Institute of Scientific and Technical Information of China (English)

    Xu-Ping Yu; Jun-Li Zhu; Xue-Ping Yao; Shi-Cheng He; Hai-Ning Huang; Wei-Liang Chen; Yong-Hao Hu; De-Bao Li


    AIM: To identify the gene (s) related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism.METHODS: Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain(s) of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame (ORF) finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used.RESULTS: A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglornerans andrimid biosynthetic gene cluster (AY192157). The Tn5was inserted at 2 420 bp of the gene corresponding to the COG3319 (the thioesterase domain of type Ⅰ polyketide synthase) coding region on B8F. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F.CONCLUSION: The anrFgene obtained is related to the antagonistic activity of B8, and the antagonistic substances produced by B8 are andrimid and/or its analogs.

  7. Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris

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    Mattanovich Diethard


    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.

  8. A biosynthetic pathway for hexanoic acid production in Kluyveromyces marxianus. (United States)

    Cheon, Yuna; Kim, Jun-Seob; Park, Jun-Bum; Heo, Paul; Lim, Jae Hyung; Jung, Gyoo Yeol; Seo, Jin-Ho; Park, Jin Hwan; Koo, Hyun Min; Cho, Kwang Myung; Park, Jin-Byung; Ha, Suk-Jin; Kweon, Dae-Hyuk


    Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.

  9. Expanding the product profile of a microbial alkane biosynthetic pathway. (United States)

    Harger, Matthew; Zheng, Lei; Moon, Austin; Ager, Casey; An, Ju Hye; Choe, Chris; Lai, Yi-Ling; Mo, Benjamin; Zong, David; Smith, Matthew D; Egbert, Robert G; Mills, Jeremy H; Baker, David; Pultz, Ingrid Swanson; Siegel, Justin B


    Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.

  10. Neurosteroid biosynthetic pathways changes in prefrontal cortex in Alzheimer's disease. (United States)

    Luchetti, Sabina; Bossers, Koen; Van de Bilt, Saskia; Agrapart, Vincent; Morales, Rafael Ramirez; Frajese, Giovanni Vanni; Swaab, Dick F


    Expression of the genes for enzymes involved in neurosteroid biosynthesis was studied in human prefrontal cortex (PFC) in the course of Alzheimer's disease (AD) (n=49). Quantitative RT-PCR (qPCR) revealed that mRNA levels of diazepam binding inhibitor (DBI), which is involved in the first step of steroidogenesis and in GABAergic transmission, were increased, as were mRNA levels for several neurosteroid biosynthetic enzymes. Aromatase, 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1) and aldo-keto reductase 1C2 (AKR1C2), were all increased in the late stages of AD. Several GABA-A subunits were significantly reduced in AD. Increased expression of aromatase in the PFC was confirmed by immunohistochemistry and was found to be localized predominantly in astrocytes. These data suggest a role for estrogens and allopregnanolone produced by astrocytes in the PFC in AD, possibly as part of a rescue program. The reduced gene expression of some synaptic and extra-synaptic GABA-A subunits may indicate a deficit of modulation of GABA-A receptors by neuroactive steroids, which may contribute to the neuropsychiatric characteristics of this disease.

  11. Antimicrobial susceptibility of Erysipelothrix rhusiopathiae isolated from pigs in Southern Japan with a modified agar dilution method. (United States)

    Chuma, Takehisa; Kawamoto, Toshio; Shahada, Francis; Fujimoto, Hideki; Okamoto, Karoku


    The determination of antimicrobial minimum inhibitory concentration (MIC) in Erysipelothrix rhusiopathiae by using the agar dilution method has not been covered by the Clinical and Laboratory Institute (CLSI). Only the broth microdilution method has been outlined. This report describes a modification of the agar dilution procedure for E. rhusiopathiae using Trypto-soy agar supplemented with 0.1% Tween 80 and incubation in ambient air at 37 degrees C for 24 hr. The MICs of the assay were in agreement with those of the broth microdilution method recommended by the CLSI. Antimicrobial susceptibility test was performed using this method for 149 E. rhusiopathiae isolates from 2 meat processing plants in Kagoshima Prefecture during the period of April 2004 to March 2005. The number of strains resistant to oxytetracycline, erythromycin, lincomycin, ofloxacin and enrofloxacin were 56 (37.6%), 4 (2.7%), 18 (12.1%), 21 (14.1%) and 19 (12.8%), respectively. All strains were susceptible to ampicillin.

  12. Biosynthetic origin of natural products isolated from marine microorganism-invertebrate assemblages. (United States)

    Simmons, T Luke; Coates, R Cameron; Clark, Benjamin R; Engene, Niclas; Gonzalez, David; Esquenazi, Eduardo; Dorrestein, Pieter C; Gerwick, William H


    In all probability, natural selection began as ancient marine microorganisms were required to compete for limited resources. These pressures resulted in the evolution of diverse genetically encoded small molecules with a variety of ecological and metabolic roles. Remarkably, many of these same biologically active molecules have potential utility in modern medicine and biomedical research. The most promising of these natural products often derive from organisms richly populated by associated microorganisms (e.g., marine sponges and ascidians), and often there is great uncertainty about which organism in these assemblages is making these intriguing metabolites. To use the molecular machinery responsible for the biosynthesis of potential drug-lead natural products, new tools must be applied to delineate their genetic and enzymatic origins. The aim of this perspective is to highlight both traditional and emerging techniques for the localization of metabolic pathways within complex marine environments. Examples are given from the literature as well as recent proof-of-concept experiments from the authors' laboratories.

  13. Polymorphism of mycotoxin biosynthetic genes among Fusarium equiseti isolates from Italy and Poland



    Fusarium equiseti (Corda) Saccardo is a soil saprophyte and a weak pathogen, associated with several diseases of fruit and other crops in subtropical and tropical areas, but also in countries with temperate climate. A wide range of secondary metabolites has been identified among natural F. equiseti populations, with zearalenone (ZEA), fusarochromanone and fusarenon-X being the most common. In present study, the genetic diversity of strains from two populations (from Italy and Poland) was eval...

  14. Proposal of a seismic isolation structure for urban tunnels and the method to evaluate its isolation effects; Shield tunnel no menshin kozo to sono menshin koka no hyoka shuho no teian

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, T. [Kumagai Gumi Co. Ltd., Tokyo (Japan); Tamura, J. [Nihon Univ., Chiba (Japan). Coll. of Industrial Technology


    An earthquake damage of a shield-driven flannel occurred during the 1985 Michoacan earthquake in the central part of Mexico City. The damages were mainly originated from tunnel axial deformations, such as tension and torsion. In order to improve seismic safety of urban tunnels due to tunnel axial deformations during earthquakes, a seismic immolation structure, in which an isolation layer is formed between a tuner lining and its surrounding soil, is presented. This paper presents a simplified method to evaluate the isolation effect of the seismic isolation structure using axisymmetric finite element models. Through parametric studies using the models, the isolation effect is evaluated in terms of the axial and torsional strain seduction ratios. Furthermore, material properties to be needed as the isolation layer is determined from static stability analyses as well as dynamic analyses. As a result, the importance of high Poisson`s ratio of the layer material is proposed. 9 refs., 15 figs., 2 tabs.

  15. Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges (United States)

    Carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV). The two cultivars exhibited different carotenoid profiles and regulatory mechanisms in flavedo and...

  16. Online RPLC-GC via TOTAD method to isolate (+)-methyl epijasmonate from lemon (Citrus limon Burm.). (United States)

    del Mar Caja, Maria; Blanch, Gracia Patricia; Ruiz del Castillo, Maria Luisa


    Pure (+)-methyl epijasmonate was isolated from lemon ( Citrus limon Burm.) for the first time. To that aim, two commercial essential oils and one homemade extract were included in the present paper. First, a study on the appropriate chromatographic conditions to avoid the epimerization from methyl epijasmonate to the more stable methyl jasmonate was accomplished. The results obtained are discussed. The presence of (+)-methyl epijasmonate in the three samples studied was initially established through the direct injection into GC-MS. However, the overlapping of (+)-methyl epijasmonate with other matrix components made it necessary to employ a multidimensional technique. RPLC-GC analysis via through-oven transfer adsorption-desorption (TOTAD) provided the selectivity and sensitivity required, reflecting that the homemade lemon extract was an adequate natural source to obtain pure (+)-methyl epijasmonate by means of the collection of the corresponding RPLC fraction.

  17. Basalt Waste Isolation Project technical program evaluation process: A criteria-based method (United States)

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

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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  19. 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


    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

  20. Chronic physical stress changes gene expression of catecholamine biosynthetic enzymes in the adrenal medulla of adult rats

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    Gavrilović Ljubica


    Full Text Available In this study we examined how chronic forced running (CFR affects the expression of catecholamine biosynthetic enzymes and cAMP response element-binding (CREB in the adrenal medulla and the weight of adrenal glands of rats. Also, we examined how CFR and additional acute immobilization stress affect the expression of catecholamine biosynthetic enzymes in the adrenal medulla and the concentration of catecholamines and corticosterone (CORT in the blood plasma. In this experiment we used as a model forced exercise in rats (treadmill running. We used the most advanced method for determining the level of gene expression, Real-time PCR with TaqMan probes, as well as Western blot analysis (ECL. We found that CFR decreases tyrosine hydroxylase (TH, and dopamine-β-hydroxylase (DBH mRNA and protein levels in the adrenal medulla. The decreased TH and DBH mRNA levels coincide with the reduced expression of CREB in the adrenal medulla and with the reduced plasma CORT level. Additionally, CFR reduces the level of phenylethanolamine N-methyltransferase (PNMT mRNA, but elevates its protein level in the adrenal medulla and increases the concentration of adrenaline (A in the plasma. Reduced level of PNMT mRNA in the adrenal medulla coincides with reduced plasma CORT level. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. The increased synthesis of PNMT enzyme in the adrenal medulla may result in an increased biosynthesis of A under chronic stress conditions. Additionally, increased level of catecholamines in the plasma after chronic physical stress is the allostatic load that may induce numerous diseases and pathological conditions.

  1. Method comparison for enhanced recovery, isolation and qualitative detection of C. jejuni and C. coli from wastewater effluent samples. (United States)

    Ugarte-Ruiz, María; Florez-Cuadrado, Diego; Wassenaar, Trudy M; Porrero, María Concepción; Domínguez, Lucas


    Seeking a sensitive protocol, culture-dependent methods were compared to detect thermophilic Campylobacter species in untreated urban effluents. We evaluated various combinations of selective media, with and without an enrichment steps, as well as an extra filtration step. Culture-independent real-time quantitative PCR was also included and all detected isolates underwent antimicrobial susceptibility testing. All tested water samples contained Campylobacter DNA, but only 64% were positive after culture. Although enrichment using Preston broth resulted in better recovery of potentially stressed Campylobacter than Bolton or Campyfood broth (CFB), there was no significant increase in efficiency compared to direct plating. The type of selective agar media used, on the other hand, had a significant effect, with CASA plates performing better than mCCDA or CFA ones. Inclusion of an enrichment step increased the ratio of C. coli vs. C. jejuni being isolated. Resistances against all antimicrobials tested were observed in C. coli, but fewer instances of resistance were found in C. jejuni isolates. Whether this difference was the result of selection during the enrichment step could not be determined. The presence of Campylobacter in urban effluents can be considered as a valuable proxy for Campylobacter populations present in urban environments.

  2. Method Comparison for Enhanced Recovery, Isolation and Qualitative Detection of C. jejuni and C. coli from Wastewater Effluent Samples

    Directory of Open Access Journals (Sweden)

    María Ugarte-Ruiz


    Full Text Available Seeking a sensitive protocol, culture-dependent methods were compared to detect thermophilic Campylobacter species in untreated urban effluents. We evaluated various combinations of selective media, with and without an enrichment steps, as well as an extra filtration step. Culture-independent real-time quantitative PCR was also included and all detected isolates underwent antimicrobial susceptibility testing. All tested water samples contained Campylobacter DNA, but only 64% were positive after culture. Although enrichment using Preston broth resulted in better recovery of potentially stressed Campylobacter than Bolton or Campyfood broth (CFB, there was no significant increase in efficiency compared to direct plating. The type of selective agar media used, on the other hand, had a significant effect, with CASA plates performing better than mCCDA or CFA ones. Inclusion of an enrichment step increased the ratio of C. coli vs. C. jejuni being isolated. Resistances against all antimicrobials tested were observed in C. coli, but fewer instances of resistance were found in C. jejuni isolates. Whether this difference was the result of selection during the enrichment step could not be determined. The presence of Campylobacter in urban effluents can be considered as a valuable proxy for Campylobacter populations present in urban environments.

  3. 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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Atsma, D.E.; Kempen, H.J.; Nieuwenhuizen, W.; van ' t Hooft, F.M.; Pauwels, E.K. (Gaubius Institute TNO, Leiden (Netherlands))


    Four {sup 99m}Tc and three {sup 123}I labeling methods were evaluated for their suitability to label low density lipoproteins (LDL) for the purpose of scintigraphic biodistribution studies. For {sup 99m}Tc these methods were: direct incorporation in LDL of {sup 99m}TcO4- using sodium dithionite (dithionite method); a method using first N,N-dimethylformamide to prepare a {sup 99m}Tc-complex reacting with LDL in a subsequent step (DMF method); a technique in which {sup 99m}TcO4- 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.

  5. 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


    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.

  6. An efficient and simple co-culture method for isolating primary human hepatic cells: Potential application for tumor microenvironment research. (United States)

    Dong, Wei; Lu, Aiguo; Zhao, Jingkun; Yin, Shuai; Ou, Baochi; Feng, Hao


    Co-cultivation of non-parenchymal cells (NPCs) and tumor cells from the same donor is important for metastatic cancer research. This study aimed to optimize a protocol for liver NPC isolation. Two novel 3D organotypic co‑culture models for hepatocyte, endothelial cell (EC) and Kupffer cell (KC) isolation were used. Long‑term cell co‑culture, density gradient centrifugation and magnetic‑activated cell sorting (MACS) were established. ECs were isolated from the co‑culture system; the purity of the ECs was 92±1.2%. The island‑like shape of hepatocytes was noted in the 3D co‑culture system, and spindle cells were found in the rest space. Immunofluorescence analysis showed a net structure; the connective tissue was positively stained with VE‑cadherin or CD68, which were ECs and KCs/macrophages. KCs were enriched in this system and separated by using selective adherence to plastic. Clec4f+ KCs consisted of 87±6.3% of these cells. Heterogeneous endothelium populations were detected, including sinusoid ECs, microvascular ECs and hepatic lymphatic vessel epithelial cells. In addition, hepatic progenitor cells were isolated and differentiated into hepatoblasts. Dendritic cells (DCs), invariant natural killer T (iNKT) cells were further separated by density gradient centrifugation and magnetic bead sorting. In the present study, high protein expression levels of desmin and GFAP were observed in the hepatic stellate cells (HSCs). Most of the HSCs were α‑SMA‑positive cells, which underlined the identity of activated HSCs. Intrahepatic human biliary epithelial cells (hBECs) were semi‑purified by centrifugation on a Percoll gradient and were further immunopurified. In conclusion, we provide an efficient long‑term culture method to obtain liver NPCs in sufficient number and purity.

  7. Rapid, sensitive and cost effective method for isolation of viral DNA from feacal samples of dogs

    Directory of Open Access Journals (Sweden)



    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

  8. Role of the hexosamine biosynthetic pathway in diabetic nephropathy. (United States)

    Schleicher, E D; Weigert, C


    The hexosamine biosynthetic pathway has been hypothesized to be involved in the development of insulin resistance and diabetic vascular complications. In particular, it was demonstrated that hyperglycemia-induced production of transforming growth factor-beta (TGF-beta1), a prosclerotic cytokine causally involved in the development of diabetic nephropathy. Several lines of evidence indicate that TGF-beta1 induction is mediated by the hexosamine pathway. In cultured mesangial cells, high glucose levels induce TGF-beta1 production. This effect is eliminated by inhibition of glutamine: fructose-6-phosphate-amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased levels of TGF-beta1 protein, mRNA, and promoter activity. Inasmuch as stimulation or inhibition of GFAT increased or decreased high glucose-stimulated activity of protein kinase C (PKC), respectively, the observed effects appear to be transduced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-alpha and basic fibroblast growth factor [bFGF]) was demonstrated in vascular smooth muscle cells. These studies also revealed a rapid increase in GFAT activity by treatment with agents that elevated levels of cyclic adenosine 3',5' monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immunohistochemistry and in situ hybridization, high expression of GFAT was found in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells. whereas glomerular cells remained essentially unstained. However, significant staining occurred in glomerular cells of patients with diabetic nephropathy. Current data indicate that the flux through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, particularly diabetic nephropathy.

  9. A new estimation method for mass of an isolated neutron star using gravitational waves

    CERN Document Server

    Ono, Kenji; Itoh, Yousuke


    We investigate a possibility of estimating mass of an isolated rapidly rotating neutron star (NS) from a continuous gravitational wave (GW) signal emitted by the NS. When the GW passes through the gravitational potential of the NS, the GW takes a slightly longer time to travel to an observer than it does in the absence of the NS. Such a time dilation effect holds also for photons and is often referred to as the gravitational time delay (or the Shapiro time delay). Correspondingly, the phase of the GW from the NS shifts due to the Coulomb type gravitational potential of the NS, and the resulting logarithmic phase shift depends on the mass, the spin frequency of the NS, and the distance to the NS. We show that the NS mass can, in principle, be obtained by making use of the phase shift difference between two modes of the continuous GW such as once and twice spin frequency modes induced by a freely precessing NS or a NS containing a pinned superfluid core. We estimate the measurement accuracy of the NS mass using...

  10. New method for the isolation of Streptococcus mutans and its differentiation from other oral streptococci. (United States)

    Linke, H A


    A new, improved agar medium for the isolation of Streptococcus mutans, the etiological agent of dental caries, was developed. In contrast to mitis-salivarius agar, this medium not only recovers a greater number of S. mutans strains from most oral specimens but, because of its mannitol and sorbitol content, it also facilitates the differentiation of S. mutans from other oral streptococci, e.g., S. salivarius, S. mitis, and S. sanguis, which do not grow or produce scanty growth only after 10 days of incubation. The medium is easy to prepare because of its simple and unique composition, is characterized by the presence of an acid indicator, and can be utilized under aerobic and anaerobic conditions as well. The medium cannot be used to distinguish among the eight serotypes, a to g and SL-1, of S. mutans. Mannitol-utilizing bacteria such as streptococci (e.g., S. faecalis) and other microorganisms (e.g., Staphylococcus aureus) are able to grow on this medium and can be distinguished from S. mutans by their unique colony morphology.

  11. [Porosity, microhardness, roughness and internal stresses of PMMA base materials depending on the isolation method. 2. Roughness and internal stresses]. (United States)

    Lockowandt, P; Loges, H; Wagner, I V


    In the present paper hot and cold polymerized denture basic materials were investigated after isolation by means of alginate, tin foil and silicone. Measurements of roughness and internal stresses after polymerization show that the advantages of a silicone isolation correspond to those of a tin foil isolation, where the clinically important smoothing effect of a silicone isolation is convincing using simple laboratory handling.

  12. 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.;


    of magnitude, (vi) we propose an approximate inference and model selection method allowing to deal with a large number of loci. We also illustrate the potential of the method by re-analyzing three datasets relative to harbour porpoises in Europe, coyotes in California and herrings in the Baltic Sea......Motivation In a recent paper, Bradburd et al. [2013] proposed a model to quantify the relative effect of geographic and environmental distance on genetic differentiation. Here, we enhance this method in several ways. Results (i) We modify the covariance model so as to fit better with mainstream...

  13. 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.;


    method allowing us to deal with genomic data sets (several hundred thousands loci). 3. We also illustrate the potential of the method by re-analysing three data sets, namely harbour porpoises in Europe, coyotes in California and herrings in the Baltic Sea. 4. The computer program developed here is freely......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...

  14. [Molecular combing method in the research of DNA replication parameters in isolated organs of Drosophyla melanogaster]. (United States)

    Ivankin, A V; Kolesnikova, T D; Demakov, S A; Andreenkov, O V; Bil'danova, E R; Andreenkova, N G; Zhimulev, I F


    Methods of physical DNA mapping and direct visualization of replication and transcription in specific regions of genome play crucial role in the researches of structural and functional organization of eukaryotic genomes. Since DNA strands in the cells are organized into high-fold structure and present as highly compacted chromosomes, the majority of these methods have lower resolution at chromosomal level. One of the approaches to enhance the resolution and mapping accuracy is the method of molecular combing. The method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends to the cover glass surface. In this article we describe the major methodological steps of molecular combing and their adaptation for researches of DNA replication parameters in polyploidy and diploid tissues of Drosophyla larvae.

  15. Identification of Candida spp. isolated from vaginal swab by phenotypic methods and multiplex PCR in Duhok, Iraq

    Directory of Open Access Journals (Sweden)

    Ahmed Basheer Mohammed


    Full Text Available Background: Candida species are the second most common cause of vulvovaginitis worldwide. The purpose of this study was to identify the species of vaginal Candida isolates by using phenotypic and Multiplex PCR techniques. Methods: 91 isolates from patients admitted to Azadi hospital and Maternity hospital in Duhok city were collected. The vaginal swab specimens were inoculated on Sabouraud dextrose agar. Colonies were then sub cultured on Chromogenic Candida agar. Genomic DNA extraction was performed using a Genomic DNA Extraction kit. For rapid identification of Candida spp., specific primers based on the genomic sequence of DNA topoisomerase 11 of C. albicans, C. parapsilosis I, C. parapsilosis II, C. guilliermondi, C. dubliniensis, C. krusei, C. kefyr and C. glabrata, C. tropicalis I, C. tropicalis II, C. lusitaniae were used. The multiplex PCR products were separated by electrophoresis in 1.5% agarose gel, visualized by staining with ethidium bromide, and photographed. Results: 4 Candida species, namely C. albicans, C. glabrata, C. krusei and C. tropicalis were distinguished by Chromogenic Candida agar on the basis of colony colour and morphology. PCR with the primer mixes yielded 7 different sized of PCR products corresponding to C. albicans, C. guilliermondii, C. dubliniensis, C. glabrata, C. kefyr, C. krusei and C. tropicalis II. The analysis revealed C. glabrata and C. albicans were the most common species isolated with the percentage 40% and 30% respectively. Conclusions: This study concluded that phenotypic characteristics on selective agar medium such as chromogenic candida agar are useful for presumptive identification of Candiada spp. with the support of molecular method such as multiplex PCR. [Int J Res Med Sci 2015; 3(11.000: 3211-3216

  16. Glucose metabolism in isolated rat adipocytes: estimate of total recovery by the product summation method. (United States)

    Groff, J L; Stugard, C E; Mays, C J; Koopmans, H S; DiGirolamo, M


    To determine the completeness of glucose recovery into its metabolic products, adipocyte glucose utilization was compared by two different methods in a basal and in an insulin-stimulated metabolic state. One of the methods (product summation) is based on the recovery of (( U-14C ))-labeled glucose in carbon dioxide and triglyceride, together with the assay of lactate and pyruvate released into the medium from the incubated cells. The second method is a modification of a procedure by which the quantity of tritiated water that is released by metabolism from +AD5-3H+BD-labeled glucose is determined. It was found that the results of the two methods were in very close agreement. Neither method measures glucose conversion to glycogen. However, glycogenic activity is very low in adipocytes and represents only a small percentage of utilized glucose. Therefore, the finding of comparable results between the two methods suggests that the summation of the metabolic products carbon dioxide, triglyceride, lactate, and pyruvate does account for nearly all of the glucose utilized. This has important implications for the understanding of the considerable variations in rate and patterns of glucose metabolism by the fat cells under a variety of experimental conditions.

  17. 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.

  18. A simple HPLC method for the isolation and quantification of the allergens tuliposide A and tulipalin A in Alstroemeria. (United States)

    Christensen, L P; Kristiansen, K


    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.

  19. Identification of Malassezia Species Isolated from Patients with Seborrheic Dermatitis Using PCR-RFLP Method in Arak, Iran

    Directory of Open Access Journals (Sweden)

    Mohajerani, HR. (PhD


    Full Text Available Background and Objective: Malassezia that is a part of normal flora is lipophilic yeast involved in a variety of skin diseases such as seborrheic dermatitis, pityriasis versicolor, atopic dermatitis and psoriasis. Seborrheic dermatitis affects most often the sebaceous-gland-rich areas of skin such as face, scalp, and parts of the upper trunk. Dandruff is a mild variant of seborrheic dermatitis characterized by scaling. In this study, Malassezia species causing dandruff were identified. Material and Methods: In this descriptive study, the samples (n= 60 from participants with dandruff were examined under a microscope using 10% KOH solution and cultured in Leeming and Notman ager medium. DNA Extraction was performed from colonies by glass bead and the Malassezia genus, and species were detected by CfoI enzyme using PCR-RFLP method Results: Of 60, 40 (66.6% were positive for Malassezia yeast. The positive samples in direct examination grew in culture medium. Malassezia species isolated were Malassezia globosa (25 cases, Malassezia restricta (10 cases, Malassezia furfur (3 cases and Malassezia sympodialis (2 cases. Conclusions: In most studies, the Malassezia species were identified as the agents causing seborrheic dermatitis. In our study, Malassezia globosa was isolated as a dominant species.

  20. Application of a DNA hybridization-hydrophobic-grid membrane filter method for detection and isolation of verotoxigenic escherichia coli. (United States)

    Todd, E C; Szabo, R A; MacKenzie, J M; Martin, A; Rahn, K; Gyles, C; Gao, A; Alves, D; Yee, A J


    Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.

  1. Application of a DNA Hybridization–Hydrophobic-Grid Membrane Filter Method for Detection and Isolation of Verotoxigenic Escherichia coli (United States)

    Todd, E. C. D.; Szabo, R. A.; MacKenzie, J. M.; Martin, A.; Rahn, K.; Gyles, C.; Gao, A.; Alves, D.; Yee, A. J.


    Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required. PMID:10543785

  2. Rural Latinos' mental wellbeing: a mixed-methods pilot study of family, environment and social isolation factors. (United States)

    Stacciarini, Jeanne-Marie R; Smith, Rebekah; Garvan, Cynthia Wilson; Wiens, Brenda; Cottler, Linda B


    Upon immigration to the rural areas in the US, Latino families may experience cultural, geographic, linguistic and social isolation, which can detrimentally affect their wellbeing by acting as chronic stressors. Using a community engagement approach, this is a pilot mixed-method study with an embedded design using concurrent qualitative and quantitative data. The purpose of this study is to evaluate family and social environments in terms of protective factors and modifiable risks associated with mental well-being in Latino immigrants living in rural areas of Florida. Latino immigrant mother and adolescent dyads were interviewed by using in-depth ethnographic semistructured interviews and subsequent quantitative assessments, including a demographic questionnaire and three structured instruments: the Family Environment Scale Real Form, the SF-12v2™ Health Survey and the short version (eight items) of PROMIS Health Organization Social Isolation. This mixed-method pilot study highlighted how family, rural, and social environments can protect or impair wellbeing in rural Latino immigrant mother and adolescent dyads.

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


    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.

  4. Effective Antibiofilm Polyketides against Staphylococcus aureus from the Pyranonaphthoquinone Biosynthetic Pathways of Streptomyces Species. (United States)

    Oja, Terhi; San Martin Galindo, Paola; Taguchi, Takaaki; Manner, Suvi; Vuorela, Pia M; Ichinose, Koji; Metsä-Ketelä, Mikko; Fallarero, Adyary


    Streptomyces bacteria are renowned for their ability to produce bioactive secondary metabolites. Recently, synthetic biology has enabled the production of intermediates and shunt products, which may have altered biological activities compared to the end products of the pathways. Here, we have evaluated the potential of recently isolated alnumycins and other closely related pyranonaphthoquinone (PNQ) polyketides against Staphylococcus aureus biofilms. The antimicrobial potency of the compounds against planktonic cells and biofilms was determined by redox dye-based viability staining, and the antibiofilm efficacy of the compounds was confirmed by viable counting. A novel antistaphylococcal polyketide, alnumycin D, was identified. Unexpectedly, the C-ribosylated pathway shunt product alnumycin D was more active against planktonic and biofilm cells than the pathway end product alnumycin A, where a ribose unit has been converted into a dioxane moiety. The evaluation of the antibiofilm potential of other alnumycins revealed that the presence of the ribose moiety in pyranose form is essential for high activity against preformed biofilms. Furthermore, the antibiofilm potential of other closely related PNQ polyketides was examined. Based on their previously reported activity against planktonic S. aureus cells, granaticin B, kalafungin, and medermycin were also selected for testing, and among them, granaticin B was found to be the most potent against preformed biofilms. The most active antibiofilm PNQs, alnumycin D and granaticin B, share several structural features that may be important for their antibiofilm activity. They are uncharged, glycosylated, and also contain a similar oxygenation pattern of the lateral naphthoquinone ring. These findings highlight the potential of antibiotic biosynthetic pathways as a source of effective antibiofilm compounds.

  5. Genome Analysis of Two Pseudonocardia Phylotypes Associated with Acromyrmex Leafcutter Ants Reveals Their Biosynthetic Potential. (United States)

    Holmes, Neil A; Innocent, Tabitha M; Heine, Daniel; Bassam, Mahmoud Al; Worsley, Sarah F; Trottmann, Felix; Patrick, Elaine H; Yu, Douglas W; Murrell, J C; Schiøtt, Morten; Wilkinson, Barrie; Boomsma, Jacobus J; Hutchings, Matthew I


    The attine ants of South and Central America are ancient farmers, having evolved a symbiosis with a fungal food crop >50 million years ago. The most evolutionarily derived attines are the Atta and Acromyrmex leafcutter ants, which harvest fresh leaves to feed their fungus. Acromyrmex and many other attines vertically transmit a mutualistic strain of Pseudonocardia and use antifungal compounds made by these bacteria to protect their fungal partner against co-evolved fungal pathogens of the genus Escovopsis. Pseudonocardia mutualists associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi make novel cyclic depsipeptide compounds called gerumycins, while a mutualist strain isolated from derived Acromyrmex octospinosus makes an unusual polyene antifungal called nystatin P1. The novelty of these antimicrobials suggests there is merit in exploring secondary metabolites of Pseudonocardia on a genome-wide scale. Here, we report a genomic analysis of the Pseudonocardia phylotypes Ps1 and Ps2 that are consistently associated with Acromyrmex ants collected in Gamboa, Panama. These were previously distinguished solely on the basis of 16S rRNA gene sequencing but genome sequencing of five Ps1 and five Ps2 strains revealed that the phylotypes are distinct species and each encodes between 11 and 15 secondary metabolite biosynthetic gene clusters (BGCs). There are signature BGCs for Ps1 and Ps2 strains and some that are conserved in both. Ps1 strains all contain BGCs encoding nystatin P1-like antifungals, while the Ps2 strains encode novel nystatin-like molecules. Strains show variations in the arrangement of these BGCs that resemble those seen in gerumycin gene clusters. Genome analyses and invasion assays support our hypothesis that vertically transmitted Ps1 and Ps2 strains have antibacterial activity that could help shape the cuticular microbiome. Thus, our work defines the Pseudonocardia species associated with Acromyrmex ants and supports the hypothesis

  6. The raman spectrum of biosynthetic human growth hormone. Its deconvolution, bandfitting, and interpretation (United States)

    Tensmeyer, Lowell G.


    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.

  7. Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous. (United States)

    Visser, Hans; van Ooyen, Albert J J; Verdoes, Jan C


    This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

  8. Isolation of four ecdysones from Ajuga iva roots and a rapid semiquantitative method for ecdysone determination. (United States)

    Sabri, N N; Asaad, A; Khafagy, S M


    The ethereal extract of AJUGA IVA (L.) S CHREB. roots, yielded 4 ecdysones. Three were proved to be cyasterone, makisterone A and ecdysterone, by using UV, IR, MS spectral methods and comparison with standard samples. Spectral data indicate that the fourth ecdysone is similar to cyasterone but having an additional hydroxy group in the side-chain. The chromatographic pattern of ecdysone content in roots, stems and leaves is presented as well as a rapid and simple Semiquantitative method for their estimation. The roots had the highest total content (0.381%).

  9. Island rescue PCR: a rapid and efficient method for isolating transcribed sequences from yeast artificial chromosomes and cosmids. (United States)

    Valdes, J M; Tagle, D A; Collins, F S


    The identification of transcripts from large genomic regions cloned in yeast artificial chromosomes (YACs) or cosmids continues to be a critical and often rate-limiting step in positional cloning of human disease genes. We have developed a PCR-based method for rapid and efficient generation of probes from YACs or cosmids that can be used for cDNA library screening. The method, which we call island rescue PCR (IRP), is based upon the observation that the 5' ends of many genes are associated with (G+C)-rich regions called CpG islands. In IRP, the YAC of interest is digested with a restriction enzyme that recognizes sequences of high CpG content, and vectorette linkers are ligated to the cleaved ends. The PCR is used to amplify the region extending from the cleaved restriction enzyme site to the nearest SINE (Alu) repeat. In many cases this product contains sequences from the 5' end of the associated gene. cDNA clones isolated with these products are then verified by mapping them back to the original YAC. The method allows rapid screening of > 500 kb of human genomic insert in one experiment, is tolerant of contaminating yeast sequences, and can also be applied to cosmid pools. In a control experiment, the method was able to identify cDNA clones for the neurofibromatosis type 1 (NF1) gene using a probe generated from a YAC in the region. Application of IRP has yielded nine other genes from YACs isolated from chromosome locations 4p16.3 and 17q21.

  10. The Prevalence of Resistance to Methicillin in Staphylococcus aureus Strains Isolated from Patients by PCR Method for Detec-tion of mecA and nuc Genes.

    Directory of Open Access Journals (Sweden)

    Roxana Sahebnasagh


    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA is the main cause of hospital infection emerged over the last decades. Rapid detection of MRSA is important for patient care and proper usage of infection control. Detection of mecA genes (encoding resistance to methicillin and other similar antibiotics and nuc genes (encoding staphylococcal thermostable nuclease by PCR method is now considered for rapid identification of MRSA strain. The aim of this study was to determine the prevalence of MRSA isolated from patients in Tehran, Iran by PCR method for detection of mecA and nuc genes.Phenotypic method such as microscopic and colony morphology and catalase and coagulase tests were used for identification of S. aureus isolates. DNA was extracted from all isolates and the presence of nuc and mecA gene was detected by PCR method. For determination of MRSA by phenotypic methods, oxacillin disk diffusion test were used. Data were analyzed by SPSS software.Out of 126 clinical sample identified by phenotypic method, 101 isolates had nuc gene. In disk diffusion tests by oxacillin disk, 78.2% of isolates were considered to be MRSA, but in PCR method for mecA gene, 69% isolates were positive.The results showed a high prevalence of methicillin-resistance among S. aureus isolates. Identifying MRSA strains, isolating MRSA-positive patients and carrier's treatment in a hospital to prevent MRSA infection is important in limiting the spread of MRSA. The PCR method for detection of nuc and mecA genes has potential for rapid and accurate diagnosis of MRSA strains.

  11. Enhanced computational methods for quantifying the effect of geographic and environmental isolation on genetic differentiation

    NARCIS (Netherlands)

    Botta, Filippo; Eriksen, Casper; Fontaine, Michael Christophe; Guillot, Gilles


    In a recent paper, Bradburd et al. (2013) proposed a model to quantify the relative effect ofgeographic and environmental distance on genetic differentiation. Here, we enhance this method in several ways. 1. We modify the covariance model so as to fit better with mainstream geostatistical models and

  12. 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.;


    Printing); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene,flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities...

  13. Cloning and characterization of the biosynthetic gene cluster of the bacterial RNA polymerase inhibitor tirandamycin from marine-derived Streptomyces sp. SCSIO1666. (United States)

    Mo, Xuhua; Wang, Zhongwen; Wang, Bo; Ma, Junying; Huang, Hongbo; Tian, Xinpeng; Zhang, Si; Zhang, Changsheng; Ju, Jianhua


    intermediates isolated from the mutants. These findings set the stage for further study of the tirandamycin biosynthetic mechanism and rationally engineer new tirandamycin analogues.

  14. Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production

    Directory of Open Access Journals (Sweden)

    Yang Haihua


    Full Text Available Abstract Background Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up nikkomycins production for further clinical trials. Results To increase the yields of nikkomycins, an additional copy of nikkomycin biosynthetic gene cluster (35 kb was introduced into nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIKwas introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of nikkomycins (880 mg L-1, 4 -fold nikkomycin X and 210 mg L-1, 1.8-fold nikkomycin Z in the resulting exconjugants comparing with the parent strain (220 mg L-1 nikkomycin X and 120 mg L-1 nikkomycin Z. The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. Conclusion A high nikkomycins producing strain (1100 mg L-1 nikkomycins was obtained by introduction of an extra nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.

  15. A unique method for isolation and solubilization of proteins after extraction of RNA from tumor tissue using trizol. (United States)

    Likhite, Neah; Warawdekar, Ujjwala M


    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.

  16. Modeling of Self-Excited Isolated Permanent Magnet Induction Generator Using Iterative Numerical Method

    Directory of Open Access Journals (Sweden)

    Mohamed Mostafa R.


    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.

  17. Method of targeted delivery of laser beam to isolated retinal rods by fiber optics. (United States)

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


    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.

  18. An efficient method for isolating antibody fragments against small peptides by antibody phage display

    DEFF Research Database (Denmark)

    Duan, Zhi; Siegumfeldt, Henrik


    We generated monoclonal scFv (single chain variable fragment) antibodies from an antibody phage display library towards three small synthetic peptides derived from the sequence of s1-casein. Key difficulties for selection of scFv-phages against small peptides were addressed. Small peptides do....... The scFvs were sequenced and characterized, and specificity was characterized by ELISA. The methods developed in this study are universally applicable for antibody phage display to efficiently produce antibody fragments against small peptides....

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

    CERN Document Server

    Sim, Nigel; Jones, C Michael; Krivitsky, Leonid


    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.

  20. Assessment of molecular methods as a tool for detecting pathogenic protozoa isolated from water bodies. (United States)

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


    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.

  1. A well-posed numerical method to track isolated conformal map singularities in Hele-Shaw flow (United States)

    Baker, Gregory; Siegel, Michael; Tanveer, Saleh


    We present a new numerical method for calculating an evolving 2D Hele-Shaw interface when surface tension effects are neglected. In the case where the flow is directed from the less viscous fluid into the more viscous fluid, the motion of the interface is ill-posed; small deviations in the initial condition will produce significant changes in the ensuing motion. This situation is disastrous for numerical computation, as small round-off errors can quickly lead to large inaccuracies in the computed solution. Our method of computation is most easily formulated using a conformal map from the fluid domain into a unit disk. The method relies on analytically continuing the initial data and equations of motion into the region exterior to the disk, where the evolution problem becomes well-posed. The equations are then numerically solved in the extended domain. The presence of singularities in the conformal map outside of the disk introduces specific structures along the fluid interface. Our method can explicitly track the location of isolated pole and branch point singularities, allowing us to draw connections between the development of interfacial patterns and the motion of singularities as they approach the unit disk. In particular, we are able to relate physical features such as finger shape, side-branch formation, and competition between fingers to the nature and location of the singularities. The usefulness of this method in studying the formation of topological singularities (self-intersections of the interface) is also pointed out.

  2. Effects of processing methods on composition and functionality of volatile components isolated from immature fruits of atemoya. (United States)

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


    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.

  3. Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts. (United States)

    Czajkowski, Robert; Ozymko, Zofia; Lojkowska, Ewa


    This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer's buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

  4. New methods for detection, analysis, and isolation of rare cell populations (United States)

    Leary, James F.; McLaughlin, Scott R.; Kavanau, Kristina S.


    New methods for flow cytometric detection, analysis and cell sorting of rare (Biplot) and commercial software packages (e.g. S-Plus for Windows). 3D stereo visualization and interactive software provide viewing of three raw and/or mathematically transformed or constructed data parameters, to aid in subsequent selection of optimal sort criteria. Special software has been developed for improved data analysis and selection of sort boundaries through the use of cell classification-tagged listmode data mixtures. This permits comparison of different classification algorithms (e.g. cluster analysis, neural networks, or recursive partitioning) for rare cells.

  5. 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


    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.

  6. Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites: preliminary characterization of its genome

    Directory of Open Access Journals (Sweden)

    Juan Carlos Garberi


    Full Text Available A rapid and simple technique for the purification of Toxoplasma gondii tachyzoites was developed. Highly purified parasites were obtained from the peritoneal exudates of infected mice by means of two consecutive discontinous sucrose gradients run at low speed (10,000xg, 30 min. Parasites obtained by this method conserved its biological activity. Hybridizations tudies with DNA from healthy mice and from purified tachyzoites preparations demonstrated that Toxoplasma gondii tachyzoites DNA could be obtained with better than 90 per cents purity. Preliminary studies with DNA endonucleases showed the presence in the tachyzoites genome of highly repetitives sequences.

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

    Directory of Open Access Journals (Sweden)

    Yulin Jia


    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.

  8. Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate. (United States)

    Manzo, Anthony J; Goushcha, Alexander O; Barabash, Yuri M; Kharkyanen, Valery N; Scott, Gary W


    Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I(exp), obeys a simple exponential law with the rate constant alphaI(exp) + k(rec), in which alpha is a parameter relating the light intensity, measured in mW/cm(2), to a corresponding theoretical rate in units of reciprocal seconds, and k(rec) is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the alpha parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer-Lambert-Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

  9. A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep. (United States)

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon; Hutmacher, Dietmar W


    The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.

  10. A facile method for isolation of recombinant human apolipoprotein A-I from E. coli. (United States)

    Ikon, Nikita; Shearer, Jennifer; Liu, Jianfang; Tran, Jesse J; Feng, ShiBo; Kamei, Ayako; Beckstead, Jennifer A; Kiss, Robert S; Weers, Paul M; Ren, Gang; Ryan, Robert O


    Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.

  11. Comparison of pulsed-field gel electrophoresis & repetitive sequence-based PCR methods for molecular epidemiological studies of Escherichia coli clinical isolates

    Directory of Open Access Journals (Sweden)

    Il Kwon Bae


    Full Text Available Background & objectives: PFGE, rep-PCR, and MLST are widely used to identify related bacterial isolates and determine epidemiologic associations during outbreaks. This study was performed to compare the ability of repetitive sequence-based PCR (rep-PCR and pulsed-field gel electrophoresis (PFGE to determine the genetic relationships among Escherichia coli isolates assigned to various sequence types (STs by two multilocus sequence typing (MLST schemes. Methods: A total of 41 extended-spectrum β-lactamase- (ESBL- and/or AmpC β-lactamase-producing E. coli clinical isolates were included in this study. MLST experiments were performed following the Achtman′s MLST scheme and the Whittam′s MLST scheme, respectively. Rep-PCR experiments were performed using the DiversiLab system. PFGE experiments were also performed. Results: A comparison of the two MLST methods demonstrated that these two schemes yielded compatible results. PFGE correctly segregated E. coli isolates belonging to different STs as different types, but did not group E. coli isolates belonging to the same ST in the same group. Rep-PCR accurately grouped E. coli isolates belonging to the same ST together, but this method demonstrated limited ability to discriminate between E. coli isolates belonging to different STs. Interpretation & conclusions: These results suggest that PFGE would be more effective when investigating outbreaks in a limited space, such as a specialty hospital or an intensive care unit, whereas rep-PCR should be used for nationwide or worldwide epidemiology studies.

  12. Comparison of Some Extraction Methods for Isolation of Catechins and Caffeine from Turkish Green Tea

    Directory of Open Access Journals (Sweden)

    Ezgi DEMİR


    Full Text Available Effective extraction of anticancer and antioxidant principles from Turkish green tea were main purpose of this work. The pre-optimized experimental condition for liquid extraction was employed for comparative appraisal.  Not only extraction methods also nature of the green tea samples (fresh, dried or frozen and quantitative yields related to collection periods were investigated.  After extraction of the green tea with various techniques the extract was partitioned with chloroform to remove caffeine, after that the extract was partitioned with ethyl acetate to obtain catechin mixture. Quantification of individual catechins was carried out by HPLC and analysis results proved that epigallocatechin gallate (EGCG was main catechin specie present in all extracts. The results indicate that hot water extraction (at 80 0C provides higher catechin yield when compared to other methods. The highest extract yields were obtained with dried leaves collected in second collection period. The crude catechin mixture contains high amount of EGCG and might be used as raw material for production of plant remedies at industrial scale.

  13. Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum. (United States)

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


    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 β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

  14. A comparative study of thermal calcination and an alkaline hydrolysis method in the isolation of hydroxyapatite from Thunnus obesus bone. (United States)

    Venkatesan, Jayachandran; Qian, Zhong Ji; Ryu, BoMi; Thomas, Noel Vinay; Kim, Se Kwon


    In the present study, hydroxyapatite (HAp) was isolated from Thunnus obesus bone using alkaline hydrolysis and thermal calcination methods. The obtained ceramic has been characterized by thermal gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), powder x-ray diffraction analysis (XRD), field-emission scanning electron microscopy, energy-dispersive x-ray analysis, transmission electron microscopy (TEM), selected area diffraction analysis, cytotoxic analysis and cell proliferation analysis. The results indicate that there are significant differences between the ceramics and T. obesus bone. FT-IR and TGA results affirmed that the collagen and organic moieties have been eliminated by both the proposed methods. XRD results were in agreement with JCPDS data. TEM and selective area diffraction images have signified that the thermal calcination method produces good crystallinity with dimensions 0.3-1.0 µm, whereas the alkaline hydrolysis method produces nanostructured HAp crystals with 17-71 nm length and 5-10 nm width. Biocompatibility of HAp crystals was evaluated by cytotoxicity and cell proliferation with human osteoblast-like cell MG-63.

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

    Directory of Open Access Journals (Sweden)

    M Thomas P Gilbert

    Full Text Available 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. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.

  16. A method for isolating piperylenes from the C5 fraction of pyrolysis gasolines

    Energy Technology Data Exchange (ETDEWEB)

    Messinger, T.; Pop, G.; Purvutolu, T.


    A method is patented for separating piperylenes from the C5 fraction of pyrolysis benzine. The starting raw material is fractionated to remove the volatile C5 components (with a boiling point of less than 38 degrees), while the remainder is fractionated, removing the upper fraction which contains piperylenes (a boiling point of 38 to 45 degrees); this fraction is subjected to extractive distillation, distilling away the residues of the C5 components; the residual product is fractionated, producing in the distillate a mixture of piperylene, acetylenes and cyclopentadienes which is shifted to the zone of extractive distillation, producing in the distillate a piperylene concentrated which is then purified by known chemical means, producing a piperylene concentrate enriched with a transisomer. The residue from the fractionation is fractionated in another tower, from the top of which the piperylene concentrated, enriched with a cis isomer is distilled off.

  17. Development of an effective and efficient DNA isolation method for Cinnamomum species. (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


    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.

  18. 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;


    , the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols....... 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...

  19. 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.

  20. 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.


    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.

  1. Hydrodistillation-adsorption method for the isolation of water-soluble, non-soluble and high volatile compounds from plant materials. (United States)

    Mastelić, J; Jerković, I; Blazević, I; Radonić, A; Krstulović, L


    Proposed method of hydrodistillation-adsorption (HDA) on activated carbon and hydrodistillation (HD) with solvent trap were compared for the isolation of water-soluble, non-soluble and high volatile compounds, such as acids, monoterpenes, isothiocyanates and others from carob (Certonia siliqua L.), rosemary (Rosmarinus officinalis L.) and rocket (Eruca sativa L.). Isolated volatiles were analyzed by GC and GC/MS. The main advantages of HDA method over ubiquitous HD method were higher yields of volatile compounds and their simultaneous separation in three fractions that enabled more detail analyses. This method is particularly suitable for the isolation and analysis of the plant volatiles with high amounts of water-soluble compounds. In distinction from previously published adsorption of remaining volatile compounds from distillation water on activated carbon, this method offers simultaneous hydrodistillation and adsorption in the same apparatus.

  2. Engineering the central biosynthetic and secondary metabolic pathways of Pseudomonas aeruginosa strain PA1201 to improve phenazine-1-carboxylic acid production. (United States)

    Jin, Kaiming; Zhou, Lian; Jiang, Haixia; Sun, Shuang; Fang, Yunling; Liu, Jianhua; Zhang, Xuehong; He, Ya-Wen


    The secondary metabolite phenazine-1-carboxylic acid (PCA) is an important component of the newly registered biopesticide Shenqinmycin. We used a combined method involving gene, promoter, and protein engineering to modify the central biosynthetic and secondary metabolic pathways in the PCA-producing Pseudomonas aeruginosa strain PA1201. The PCA yield of the resulting strain PA-IV was increased 54.6-fold via the following strategies: (1) blocking PCA conversion and enhancing PCA efflux pumping; (2) increasing metabolic flux towards the PCA biosynthetic pathway through the over-production of two DAHP synthases and blocking the synthesis of 21 secondary metabolites; (3) increasing the PCA precursor supply through the engineering of five chorismate-utilizing enzymes; (4) engineering the promoters of two PCA biosynthetic gene clusters. Strain PA-IV produced 9882 mg/L PCA in fed-batch fermentation, which is twice as much as that produced by the current industrial strain. Strain PA-IV was also genetically stable and comparable to Escherichia coli in cytotoxicity.

  3. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli. (United States)

    Stahlhut, Steen G; Siedler, Solvej; Malla, Sailesh; Harrison, Scott J; Maury, Jérôme; Neves, Ana Rute; Forster, Jochen


    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer's disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin pathway in Escherichia coli. We propose a novel biosynthetic pathway from the amino acid, tyrosine, utilizing nine heterologous enzymes. The pathway proceeds via the synthesis of two flavanones never produced in microorganisms before--garbanzol and resokaempferol. We show for the first time a functional biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols.

  4. Volatile terpenes from actinomycetes: a biosynthetic study correlating chemical analyses to genome data. (United States)

    Rabe, Patrick; Citron, Christian A; Dickschat, Jeroen S


    The volatile terpenes of 24 actinomycetes whose genomes have been sequenced (or are currently being sequenced) were collected by use of a closed-loop stripping apparatus and identified by GC/MS. The analytical data were compared against a phylogenetic analysis of all 192 currently available sequences of bacterial terpene cyclases (excluding geosmin and 2-methylisoborneol synthases). In addition to the several groups of terpenes with known biosynthetic origin, selinadienes were identified as a large group of biosynthetically related sesquiterpenes that are produced by several streptomycetes. The detection of a large number of previously unrecognised side products of known terpene cyclases proved to be particularly important for an in depth understanding of biosynthetic pathways to known terpenes in actinomycetes. Interpretation of the chemical analytical data in the context of the phylogenetic tree of bacterial terpene cyclases pointed to the function of three new enzymes: (E)-β-caryophyllene synthase, selina-3,7(11)-diene synthase and aristolochene synthase.

  5. 甜菜根腐病病原分离方法研究%Study on isolation methods of sugarbeet root rot pathogens

    Institute of Scientific and Technical Information of China (English)

    刘浩; 张蓉


    By adopting inducing isolation and usual tissue isolation,it proves that the inducing isolation is a good method to isolate pathogens which can produce zoospore such as Phytophthora and Pythium etc.%通过采用诱导分离法和常规组织分离法,对甜菜根腐病病原研究结果证明,诱导法更有利于对Phytophthora、 Pythium等可产生游动孢子的病原菌的分离。

  6. 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


    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.

  7. Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes. (United States)

    Horsman, Geoff P; Chen, Yihua; Thorson, Jon S; Shen, Ben


    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.

  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


    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. 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


    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.

  10. 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


    considerable diversity: only 2% of the chemical features and 7% of the biosynthetic genes were common to all strains, while 30% of all features and 24% of the genes were unique to single strains. The list of chemical features was reduced to 50 discriminating features using a genetic algorithm and support...... vector machines. Features were dereplicated by tandem mass spectrometry (MS/MS) networking to identify molecular families of the same biosynthetic origin, and the associated pathways were probed using comparative genomics. Most of the discriminating features were related to antibacterial compounds...

  11. Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli

    DEFF Research Database (Denmark)

    Stahlhut, Steen Gustav; Siedler, Solvej; Malla, Sailesh;


    Plant secondary metabolites are an underutilized pool of bioactive molecules for applications in the food, pharma and nutritional industries. One such molecule is fisetin, which is present in many fruits and vegetables and has several potential health benefits, including anti-cancer, anti......-viral and anti-aging activity. Moreover, fisetin has recently been shown to prevent Alzheimer׳s disease in mice and to prevent complications associated with diabetes type I. Thus far the biosynthetic pathway of fisetin in plants remains elusive. Here, we present the heterologous assembly of a novel fisetin...... biosynthetic pathway and establish E. coli as a microbial platform strain for the production of fisetin and related flavonols....

  12. A quick, efficient, and cost-effective method for isolating high-quality total RNA from tomato fruits, suitable for molecular biology studies. (United States)

    Sabzevari, Alireza Ghannad; Hosseini, Ramin


    Tomato (Solanum lycopersicum L.) is the primary model for the study of fleshy fruits, and research on this species has elucidated many aspects of fruit physiology, development, and metabolism. However, for advancing such studies at molecular biology levels, the RNA isolation from fruit tissues is often essential. The RNA isolation from tomato fruits is complicated because of the presence of high levels of polysaccharides, polyphenolics, pigments, and secondary metabolites and also the varying water content during development. Here, we present an optimized protocol for the isolation of total RNA from the fruit tissues at different developmental stages. In comparison to the previous methods described for the RNA isolation from tomato fruit, this method has the advantages that it does not involve the use of guanidine salts, lyophilizers, and commercial reagents, reduces the time and cost of extraction, overcomes the high water content problem, and promotes RNA quality by inhibiting RNA degradation and minimizing the gDNA, polyphenolic and polysaccharide contaminations. Using this method, high yields of high-purity and intact RNA samples were obtained as confirmed by the spectrophotometric readings and the electrophoresis on denaturing agarose gels. The isolated RNA was employed as a robust template for cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), and temporal gene expression analysis. The functionality of the isolated RNA was further demonstrated through cloning full-length cDNAs encoding β-galactosidase proteins by RT-PCR and sequencing.

  13. Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection.

    Directory of Open Access Journals (Sweden)

    Nao Sugiyama

    Full Text Available The hepatitis C virus (HCV culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.

  14. Comparison of subtypes of Listeria monocytogenes isolates from naturally contaminated watershed samples using a combination of non-selective and selective enrichment methods (United States)

    Two enrichment methods for Listeria monocytogenes using Immuno Magnetic Separation were tested to determine if they selected the same subtypes of isolates. Both methods included a non-selective enrichment and one included subculture in Fraser Broth. Naturally contaminated watershed samples from the ...

  15. 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.


    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

  16. Influence of Bovine Whey Protein Concentrate and Hydrolysate Preparation Methods on Motility in the Isolated Rat Distal Colon (United States)

    Dalziel, Julie E.; Anderson, Rachel C.; Bassett, Shalome A.; Lloyd-West, Catherine M.; Haggarty, Neill W.; Roy, Nicole C.


    Whey protein concentrate (WPC) and hydrolysate (WPH) are protein ingredients used in sports, medical and pediatric formulations. Concentration and hydrolysis methods vary for whey sourced from cheese and casein co-products. The purpose of this research was to investigate the influence of whey processing methods on in vitro gastrointestinal (GI) health indicators for colonic motility, epithelial barrier integrity and immune modulation. WPCs from casein or cheese processing and WPH (11% or 19% degree of hydrolysis, DH) were compared for their effects on motility in a 1 cm section of isolated rat distal colon in an oxygenated tissue bath. Results showed that WPC decreased motility irrespective of whether it was a by-product of lactic acid or mineral acid casein production, or from cheese production. This indicated that regardless of the preparation methodology, the whey protein contained components that modulate aspects of motility within the distal colon. WPH (11% DH) increased contractile frequency by 27% in a delayed manner and WPH (19% DH) had an immediate effect on contractile properties, increasing tension by 65% and frequency by 131%. Increased motility was associated with increased hydrolysis that may be attributed to the abundance of bioactive peptides. Increased frequency of contractions by WPH (19% DH) was inhibited (by 44%) by naloxone, implicating a potential involvement of opioid receptors in modulation of motility. Trans-epithelial electrical resistance and cytokine expression assays revealed that the WPC proteins studied did not alter intestinal barrier integrity or elicit any discernible immune response. PMID:27983629

  17. Isolation of αL I domain mutants mediating firm cell adhesion using a novel flow-based sorting method. (United States)

    Pepper, Lauren R; Parthasarathy, Ranganath; Robbins, Gregory P; Dang, Nicholas N; Hammer, Daniel A; Boder, Eric T


    The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.

  18. Intracloacal inoculation, an effective screening method for determining the efficacy of probiotic bacterial isolates against Campylobacter colonization in broiler chickens. (United States)

    Arsi, K; Donoghue, A M; Woo-Ming, A; Blore, P J; Donoghue, D J


    Campylobacter is a leading cause of foodborne illness worldwide. It is common in poultry, and human infections are often associated with consumption of contaminated poultry products. One strategy to reduce Campylobacter colonization in poultry is the use of oral probiotics, but this produces variable results, possibly because the probiotics are destroyed in the stomach's acidic environment. Protection (e.g., encapsulation) of isolates may overcome this problem, but there is no assurance that these isolates will have efficacy in the lower gastrointestinal tract. Therefore, screening candidate isolates by directly placing them in the lower intestinal tract via cloacal inoculation may eliminate the time and expense of encapsulating ineffective isolates. Thus, the purpose of this study was to collect bacterial isolates with anti-Campylobacter activity in vitro and evaluate their efficacy in vivo upon either oral or intracloacal administration. Bacterial isolates were collected from healthy birds and were evaluated for efficacy against C. jejuni in vitro. Isolates having generally regarded as safe status and demonstrating in vitro anti-Campylobacter properties were evaluated after oral or intracloacal inoculation into chicks on day 1 (n = 10 birds per isolate per route of administration). On day 7, birds were dosed by oral gavage with a four-strain mixture of wild-type Campylobacter containing at least 1 × 10(7) CFU/ml organisms. On day 14, birds were euthanized and the ceca were collected aseptically for Campylobacter enumeration. When dosed orally, only one isolate had a 1-log reduction in cecal Campylobacter counts, whereas when administered intracloacally, six of these isolates produced a 1- to 3-log reduction in cecal Campylobacter counts in 14-day-old chickens. These results support the strategy of evaluating the efficacy of potential probiotic isolates via cloacal inoculation prior to undergoing the effort of encapsulating isolates for oral administration.

  19. Effects of overexpressing individual lignin biosynthetic enzymes on feeding and growth of corn earworms and fall armyworms (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...

  20. Complete Genome Sequence of the Filamentous Fungus Aspergillus westerdijkiae Reveals the Putative Biosynthetic Gene Cluster of Ochratoxin A (United States)

    Chakrabortti, Alolika; Li, Jinming


    Ochratoxin A (OTA) is a common mycotoxin that contaminates food and agricultural products. Sequencing of the complete genome of Aspergillus westerdijkiae, a major producer of OTA, reveals more than 50 biosynthetic gene clusters, including a putative OTA biosynthetic gene cluster that encodes a dozen of enzymes, transporters, and regulatory proteins. PMID:27635003

  1. 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


    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)

  2. Natural product proteomining, a quantitative proteomics platform, allows rapid discovery of biosynthetic gene clusters for different classes of natural products. (United States)

    Gubbens, Jacob; Zhu, Hua; Girard, Geneviève; Song, Lijiang; Florea, Bogdan I; Aston, Philip; Ichinose, Koji; Filippov, Dmitri V; Choi, Young H; Overkleeft, Herman S; Challis, Gregory L; van Wezel, Gilles P


    Information on gene clusters for natural product biosynthesis is accumulating rapidly because of the current boom of available genome sequencing data. However, linking a natural product to a specific gene cluster remains challenging. Here, we present a widely applicable strategy for the identification of gene clusters for specific natural products, which we name natural product proteomining. The method is based on using fluctuating growth conditions that ensure differential biosynthesis of the bioactivity of interest. Subsequent combination of metabolomics and quantitative proteomics establishes correlations between abundance of natural products and concomitant changes in the protein pool, which allows identification of the relevant biosynthetic gene cluster. We used this approach to elucidate gene clusters for different natural products in Bacillus and Streptomyces, including a novel juglomycin-type antibiotic. Natural product proteomining does not require prior knowledge of the gene cluster or secondary metabolite and therefore represents a general strategy for identification of all types of gene clusters.

  3. 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


    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.

  4. Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

    NARCIS (Netherlands)

    El Khattabi, Mohamed; van Roosmalen, Maarten L.; Jager, Dennis; Metselaar, Heidi; Permentier, Hjalmar; Leenhouts, Kees; Broos, Jaap


    Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorporation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is th



    Dan Cascaval; Anca-Irina Galaction


    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.

  6. Sugars as the optimal biosynthetic carbon substrate of aqueous life throughout the universe (United States)

    Weber, A. L.


    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.

  7. Phytochemical and Biosynthetic Studies of Lignans, with a Focus on Indonesian Medicinal Plants

    NARCIS (Netherlands)

    Elfahmi, [No Value


    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. 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...

  9. 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


    [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.

  10. 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


    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.

  11. Detection of Ampicillin Resistance Genes (bla in Clinical Isolates of Escherichia coli with Polymerase Chain Reaction Method

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    Tiana Milanda


    Full Text Available Escherichia coli is a rod negative Gram which could be pathogenic, if its value increases or located in outer gastrointestinal tract. Pathogenic E. coli will produce enterotoxin which will cause diarrhoea or infection in urine tract. Ampicilin was one of particular antibiotics to overcome infection. Ampicilin nowadays is no longer used as primary medicine, because of its resistance case. The aim of this research is to detect the presence of gene which is responsible to ampicilin resistant E. coli. We used isolated midstream urine from cystitis object in Hasan Sadikin Hospital (RSHS as samples. Polymerase Chain Reaction (PCR method (colony-PCR and DNA-PCR were done to invenstigate the antibiotic resistency. Based on the result of antibiotic susceptibility testing to ampicillin, E. coli samples were resistant to ampicilin. Elektroforegram products of colony-PCR and DNA-PCR showed that the resistance case of ampicilin caused by bla gene (199 bp. Selective and rational antibiotic treatment is required to prevent ampicillin resistance in patients with symptoms

  12. Isolation of a new class of ecdysteroid conjugates (glucosyl-ferulates) using a combination of liquid chromatographic methods. (United States)

    Ho, Raimana; Girault, Jean-Pierre; Cousteau, Pierre-Yves; Bianchini, Jean-Pierre; Raharivelomanana, Phila; Lafont, René


    The Polynesian medicinal fern Microsorum membranifolium contains very large amounts of ecdysteroids, including ecdysone, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and 2-deoxyecdysone. It also contains large amounts of unusual ecdysteroids which have been unambiguously identified by mass spectrometry and nuclear magnetic resonance. A new class of ecdysteroid conjugates (3-glucosyl-ferulates of 2-deoxyecdysone and 2-deoxy-20-hydroxyecdysone) is isolated, together with a new glycoside (2-deoxyecdysone 25-rhamnoside). The simultaneous presence of a sugar and an aromatic moiety results in a very particular chromatographic behavior of these conjugates. They behave like flavonoids and polyphenols when using the classical purification on polyamide, aimed at removing the latter from crude plant extracts, and would therefore be lost. They elute as non-polar ecdysteroids on reversed-phase high-performance liquid chromatography (RP-HPLC), whereas their behavior on normal-phase (NP) HPLC is strongly dependent on the mobile phase composition. Our data highlight the importance of selectivity in the choice of HPLC methods used for ecdysteroid separations.

  13. Assessment of telavancin minimal inhibitory concentrations by revised broth microdilution method in phase 3 complicated skin and skin-structure infection clinical trial isolates. (United States)

    Smart, Jennifer I; Corey, G Ralph; Stryjewski, Martin E; Wang, Whedy; Barriere, Steven L


    The broth microdilution (BMD) MIC testing method for telavancin was recently revised BMD (rBMD) to improve accuracy and reproducibility. Staphylococcus aureus isolates from telavancin phase 3 complicated skin and skin-structure infection (cSSSI) studies were tested using the rBMD method. Retesting of 1132 isolates produced MICs ranging from ≤0.015 to 0.12μg/mL that were 8-fold lower than the original method. All isolates tested remained susceptible to telavancin at the revised susceptibility breakpoint of 0.12μg/mL. The clinical cure and microbiological eradication rates were 90% (368/409) and 89% (366/409) for telavancin-treated patients, and were similar for patients with methicillin-susceptible and -resistant S. aureus isolates and S. aureus isolates with elevated vancomycin MICs (≥1μg/mL). The data presented here are aimed to update the literature and better inform clinicians and clinical microbiologists about the revised telavancin MICs, as well as the corresponding clinical and microbiological cure rates for cSSSI patients.

  14. Interlaboratory Study of Quality Control Isolates for a Broth Microdilution Method (Modified CLSI M38-A) for Testing Susceptibilities of Dermatophytes to Antifungals▿ (United States)

    Ghannoum, M. A.; Arthington-Skaggs, B.; Chaturvedi, V.; Espinel-Ingroff, A.; Pfaller, M. A.; Rennie, R.; Rinaldi, M. G.; Walsh, T. J.


    The Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, or NCCLS) M38-A standard for the susceptibility testing of filamentous fungi does not specifically address the testing of dermatophytes. In 2003, a multicenter study investigated the reproducibility of the microdilution method developed at the Center for Medical Mycology, Cleveland, Ohio, for testing the susceptibility of dermatophytes. Data from that study supported the introduction of this method for testing dermatophytes in the future version of the CLSI M38-A standard. In order for the method to be accepted by CLSI, appropriate quality control isolates needed to be identified. To that end, an interlaboratory study, involving the original six laboratories plus two additional sites, was conducted to evaluate potential candidates for quality control isolates. These candidate strains included five Trichophyton rubrum strains known to have elevated MICs to terbinafine and five Trichophyton mentagrophytes strains. Antifungal agents tested included ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Based on the data generated, two quality control isolates, one T. rubrum isolate and one T. mentagrophytes isolate, were identified and submitted to the American Type Culture Collection (ATCC) for inclusion as reference strains. Ranges encompassing 95.2 to 97.9% of all data points for all seven drugs were established. PMID:17050812

  15. Profile of antimicrobial susceptibility isolated microorganisms from hospitalized patients in PICU ward and detection of Methicillin-resistant Staphylococcus aureus and ESBL-producing bacteria by phenotypic methods

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    Shahla Abbas Poor


    Full Text Available Background: Hospital-acquired infections are a major challenge to patient. A range of gram-negative organisms are responsible for hospital-acquired infections, the Enterobacteriaceae family being the most commonly identified group overall. Infections by ESBL producers are associated with severe adverse clinical outcomes that have led to increased mortality, prolonged hospitalization, and rising medical costs. The aim of this study was to survey profile of antimicrobial susceptibility isolated microorganisms from hospitalized patients in PICU ward and detection of methicillin-resistant Staphylococcus aureus and ESBL-producing bacteria by phenotypic methods. Material and Methods: In this study participants were patients hospitalized in PICU part of Bahrami Hospital, Tehran, with attention to involved organ. For isolation of bacteria from patient’s samples, culture performed on different selective and differential media. After confirmation of bacteria by biochemical tests, susceptibility testing was performed by disc diffusion method. Phenotypic detection of MRSA strains was performed using cefoxcitin disc. ESBL producing strains were detected by ceftazidime (CAZ and ceftazidime/clavulanic acid (CAZ/CLA discs. Results: Among all isolated organisms from clinical samples, the most common isolated organisms were Escherichia coli (24 cases, Pseudomonas areoginosa (9 cases and Staphylococcus aureus (8 cases, respectively. Among eight MRSA isolated strains from different clinical samples, six strains (75% were MRSA. Among 52 isolated gram negative organisms, 5 strains (9/6% were ESBL. Conclusion: Standard interventions to prevent the transmission of antimicrobial resistance in health care facilities include hand hygiene, using barrier precautions in the care of colonized and infected patients, using dedicated instruments and equipment for these patients. The colonized or infected patients should be isolated in single rooms, multibed rooms or areas

  16. Characterization of the CDP-D-mannitol biosynthetic pathway in Streptococcus pneumoniae 35A. (United States)

    Wang, Quan; Xu, Yanli; Perepelov, Andrei V; Knirel, Yuriy A; Reeves, Peter R; Shashkov, Alexander S; Ding, Peng; Guo, Xi; Feng, Lu


    Streptococcus pneumoniae is a major human pathogen associated with diseases worldwide. The capsular polysaccharides (CPSs) are considered a major virulence factor and are targets for a vaccine. d-Mannitol was found to be present in the CPS of several S. pneumoniae serotypes. Two genes, mnp1 and mnp2, which are located in the CPS gene cluster, were proposed to be responsible for the synthesis of NDP-d-mannitol (the nucleotide activated form of d-mannitol). However, the pathway has never been identified by experimental methods and we aimed to characterize it in the present study. To achieve this, the two genes, mnp1 and mnp2, were cloned and the gene products were overexpressed, purified, and analyzed in vitro for their respective enzymatic activities. Products of reactions catalyzed by Mnp1 and Mnp2 were detected by capillary electrophoresis and validated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. We show that Mnp1 is responsible for the transfer of CMP from CTP to d-fructose-6-phosphate (Fru-6-P) to form CDP-d-fructose, whereas Mnp2 catalyzed the conversion of CDP-d-fructose to CDP-d-mannitol. Therefore, Mnp1 (renamed as mnpA) was identified as Fru-6-P cytidylyltransferase-encoding gene, and mnp2 (renamed as mnpB) as a CDP-d-fructose reductase-encoding gene. The kinetics of Mnp1 for the substrate (Fru-6-P and CTP) and of Mnp2 for the substrate (CDP-d-fructose) and the cofactor NADH or NADPH fitted the Michaelis-Menten model. The effects of temperature, pH and cations on the two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-d-mannitol has been identified biochemically.

  17. The Sound of Silence: Activating Silent Biosynthetic Gene Clusters in Marine Microorganisms

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    F. Jerry Reen


    Full Text Available Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs. However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

  18. Kinetic model of metabolic network for xiamenmycin biosynthetic optimisation. (United States)

    Xu, Min-juan; Chen, Yong-cong; Xu, Jun; Ao, Ping; Zhu, Xiao-mei


    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.

  19. A comparison study of the EF-18 agar/Hydrophobic Grid Membrane Filter (HGMF) method and the enzyme linked antibody (ELA)/HGMF method to the HPB standard method in the isolation of Salmonella. (United States)

    Warburton, D W; Arling, V; Worobec, S; Mackenzie, J; Todd, E C; Lacasse, P; Lamontagne, G; Plante, R; Shaw, S; Bowen, B


    As part of a comparative and collaborative study of rapid methods for the detection of Salmonella and the standard Health Protection Branch (HPB) method, six Federal and Provincial Laboratories compared the EF-18 agar/Hydrophobic Grid Membrane Filters (HGMF) method to the standard HPB method. Two Federal Laboratories also compared the enzyme linked antibody (ELA)/HGMF method (which is a further development of the EF-18 agar/HGMF method) to the standard method. During this study the false-negative rates ranged from 0% to 15% for the standard HPB method, from 5.88% to 43.5% for the EF-18 agar/HGMF method, and from 0% to 10.5% for the ELA/HGMF method. The EF-18 agar/HGMF method did not compare well with the standard HPB method due to the number of false-negatives. Problems with this method resulted from the inability to isolate colonies of Salmonella on the HGMF due to the small colony size, abnormal colony coloration, and overgrowth by competitors. The ELA/HGMF method, however, was shown to be comparable to the standard HPB method. The main advantages of this method are that the antibody-staining step is independent of colony coloration and carbohydrate utilization on the plating media; the ability to detect some unusual strains of Salmonella irrespective of their atypical reactions on the media; and the ELA staining can indicate the presence of Salmonella even when the HGMF is overgrown by competitors. Also, cultural confirmation can proceed simultaneously yet independently of the ELA staining procedure. The data presented here indicate that this method is worth further study.

  20. Characterization of the biosynthetic gene cluster of rebeccamycin from Lechevalieria aerocolonigenes ATCC 39243. (United States)

    Onaka, Hiroyasu; Taniguchi, Shin-ichi; Igarashi, Yasuhiro; Furumai, Tamotsu


    The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2 disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). rebT disruptants produced rebeccamycin as much as the wild strain, so rebT was probably not involved in rebeccamycin production. Biosynthetic genes of staurosporine, an another indolocarbazole antibiotic, were cloned from Streptomyces sp. TP-A0274. staO, staD, and staP were similar to rebO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes.

  1. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10. (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


    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.

  2. Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

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    Honghao Chen


    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.

  3. Targeted Gene Disruption of the Cyclo (L-Phe, L-Pro Biosynthetic Pathway in Streptomyces sp. US24 Strain

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    Samiha Sioud


    Full Text Available We have previously isolated a new actinomycete strain from Tunisian soil called Streptomyces sp. US24, and have shown that it produces two bioactive molecules including a Cyclo (L-Phe, L-Pro diketopiperazine (DKP. To identify the structural genes responsible for the synthesis of this DKP derivative, a PCR amplification (696 bp was carried out using the Streptomyces sp. US24 genomic DNA as template and two degenerate oligonucleotides designed by analogy with genes encoding peptide synthetases (NRPS. The detection of DKP derivative biosynthetic pathway of the Streptomyces sp. US24 strain was then achieved by gene disruption via homologous recombination using a suicide vector derived from the conjugative plasmid pSET152 and containing the PCR product. Chromatography analysis, biological tests and spectroscopic studies of supernatant cultures of the wild-type Streptomyces sp. US24 strain and three mutants obtained by this gene targeting disruption approach showed that the amplified DNA fragment is required for Cyclo (L-Phe, L-Pro biosynthesis in Streptomyces sp. US24 strain. This DKP derivative seems to be produced either directly via a nonribosomal pathway or as a side product in the course of nonribosomal synthesis of a longer peptide.

  4. Characterization of the De Novo Biosynthetic Enzyme of Platelet Activating Factor, DDT-Insensitive Cholinephosphotransferase, of Human Mesangial Cells

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    Alexandros Basilios Tsoupras


    Full Text Available Platelet activating factor (PAF, a potent inflammatory mediator, is implicated in several proinflammatory/inflammatory diseases such as glomerulonephritis, glomerulosclerosis, atherosclerosis, cancer, allergy, and diabetes. PAF can be produced by several renal cells under appropriate stimuli and it is thought to be implicated in renal diseases. The aim of this study is the characterization of DTT-insensitive cholinephosphotransferase (PAF-CPT of human mesangial cell (HMC, the main regulatory enzyme of PAF de novo biosynthetic pathway. Microsomal fractions of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by TLC and in vitro biological test in rabbit washed platelets. The effect of bovine serum albumin (BSA, dithiothreitol (DTT, divalent cations (Mg2+ and Ca2+, EDTA, and various chemicals on the activity of PAF-CPT of HMC was also studied. Moreover, preliminary in vitro tests have been performed with several anti-inflammatory factors such as drugs (simvastatin, IFNa, rupatadine, tinzaparin, and salicylic acid and bioactive compounds of Mediterranean diet (resveratrol and lipids of olive oil, olive pomace, sea bass “Dicentrarchus labrax,” and gilthead sea bream “Sparus aurata”. The results indicated that the above compounds can influence PAF-CPT activity of HMC.

  5. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. (United States)

    Harashima, S; Hinnebusch, A G


    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

  6. Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq

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    Xiasheng Zheng


    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.

  7. Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer Streptomyces collinus Tü 365. (United States)

    Iftime, Dumitrita; Kulik, Andreas; Härtner, Thomas; Rohrer, Sabrina; Niedermeyer, Timo Horst Johannes; Stegmann, Evi; Weber, Tilmann; Wohlleben, Wolfgang


    Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC-MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.

  8. Isolation of Inositol Hexaphosphate (IHP)-Degrading Bacteria from Arbuscular Mycorrhizal Fungal Hyphal Compartments Using a Modified Baiting Method Involving Alginate Beads Containing IHP (United States)

    Hara, Shintaro; Saito, Masanori


    Phytate (inositol hexaphosphate; IHP)-degrading microbes have been suggested to contribute to arbuscular mycorrhizal fungi (AMF)-mediated P transfer from IHP to plants; however, no IHP degrader involved in AMF-mediated P transfer has been isolated to date. We herein report the isolation of IHP-degrading bacteria using a modified baiting method. We applied alginate beads as carriers of IHP powder, and used them as recoverable IHP in the AM fungal compartment of plant cultivation experiments. P transfer from IHP in alginate beads via AMF was confirmed, and extracted DNA from alginate beads was analyzed by denaturing gradient gel electrophoresis targeting the 16S rRNA gene and a clone library method for the beta-propeller phytase (BPP) gene. The diversities of the 16S rRNA and BPP genes of microbes growing on IHP beads were simple and those of Sphingomonas spp. and Caulobacter spp. dominated. A total of 187 IHP-utilizing bacteria were isolated and identified, and they were consistent with the results of DNA analysis. Furthermore, some isolated Sphingomonas spp. and Caulobacter sp. showed IHP-degrading activity. Therefore, we successfully isolated dominant IHP-degrading bacteria from IHP in an AMF hyphal compartment. These strains may contribute to P transfer from IHP via AMF. PMID:27383681

  9. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae. (United States)

    He, Yanxia; Guo, Xianguang; Xiang, Shifei; Li, Jiao; Li, Xiaoqin; Xiang, Hui; He, Jinlei; Chen, Dali; Chen, Jianping


    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.

  10. Identification of Echinococcus Granulosus Strains in Isolated Hydatid Cyst Specimens from Animals by PCR-RFLP Method in West Azerbaijan – Iran

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    Haleh Hanifian


    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

  11. An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke. (United States)

    Dalrymple, Annette; Ordoñez, Patricia; Thorne, David; Dillon, Debbie; Meredith, Clive


    Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies.

  12. A user friendly method to isolate and single spore the fungi Magnaporthe oryzae and Magnaporthe grisea obtained from diseased field samples (United States)

    The fungus Magnaporthe oryzae is the causal agent for a wide range of plant diseases including diseases of rice, wheat, rye grass, turfgrass and pearl millet. A simple robust procedure for fungal isolation is not publicly available. In the present study, a user friendly method was developed to iso...

  13. Evaluation of Etest and macrodilution broth method for antifungal susceptibility testing of Candida sp strains isolated from oral cavities of AIDS patients

    Directory of Open Access Journals (Sweden)

    SILVA Maria do Rosário R.


    Full Text Available A comparison of the Etest and the reference broth macrodilution susceptibility test for fluconazole, ketoconazole, itraconazole and amphotericin B was performed with 59 of Candida species isolated from the oral cavities of AIDS patients. The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines. Our data showed that there was a good correlation between the MICs obtained by the Etest and broth dilution methods. When only the MIC results at ± 2 dilutions for both methods were considered, the agreement rates were 90.4% for itraconazole, ketoconazole and amphotericin B and 84.6% for fluconazole of the C. albicans tested. In contrast, to the reference method, the Etest method classified as susceptible three fluconazole-resistant isolates and one itraconazole-resistant isolate, representing four very major errors. These results indicate that Etest could be considered useful for antifungal sensitivity evaluation of yeasts in clinical laboratories.

  14. Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201. (United States)

    Peng, Linda X; Wallace, Morgan; Andaloro, Bridget; Fallon, Dawn; Fleck, Lois; Delduco, Dan; Tice, George


    The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.

  15. Detection of KPC Carbapenemase in Pseudomonas aeruginosa Isolated From Clinical Samples Using Modified Hodge Test and Boronic Acid Phenotypic Methods and Their Comparison With the Polymerase Chain Reaction (United States)

    Falahat, Saeed; Shojapour, Mana; Sadeghi, Abdorrahim


    Background Bacterial resistance to antibiotics has become a major source of concern for public health. Pseudomonas aeruginosa strains are important opportunistic pathogens. These bacteria have a high resistance to a wide range of existing antimicrobials and antibiotics. Objectives The present study was performed to evaluate the frequency of KPC in P. aeruginosa isolated from clinical samples of educational hospitals of Arak University of Medical Sciences, using the mentioned phenotypic and genotypic methods. Materials and Methods One hundred and eight non-duplicate clinical isolates of P. aeruginosa were collected from hospitals of Arak University of Medical Sciences, Arak, Iran. Antibacterial susceptibility was determined by the disk diffusion method. KPC production was confirmed by the Modified Hodge Test (MHT), which is a phenotypic test, and combined-disk test with boronic acid and the Polymerase Chain Reaction (PCR). Results In the present study, 13 isolates (12%) of P. aeruginosa were positive for KPC, using PCR. Comparison of the two phenotypic methods used in this study showed that boronic acid is more sensitive than MHT in identification of KPC-producing strains (84.6% vs. 77%). Conclusions Utilization of reliable methods for identifying carbapenemase-producing strains and determining their antibiotic resistance pattern could have a very important role in treatment of infections caused by these strains. A substantial amount of P. aeruginosa isolated from clinical samples of hospitals in Arak (Iran) produce KPC carbapenemase. Due to their low specificity, MHT and boronic acid phenotypic methods could not completely identify KPC-producing P. aeruginosa. However, the sensitivity of boronic acid phenotypic method in detection of KPC was higher than MHT.

  16. A new method for evaluating flowering synchrony to support the temporal isolation of genetically modified crops from their wild relatives. (United States)

    Ohigashi, Kentaro; Mizuguti, Aki; Yoshimura, Yasuyuki; Matsuo, Kazuhito; Miwa, Tetsuhisa


    Hybridization between crops and their wild relatives potentially threatens the genetic identity of the wild plants, particularly in the case of genetically modified crops. Only a few studies have examined the use of temporal isolation to prevent hybridization, and the indices used in those studies, (e.g., the days of flowering overlap), are not precise to evaluate the degree of synchrony in flowering. Here we propose a flowering similarity index that can compare the degree of flowering synchrony between two relevant species and measure the efficiency of temporal isolation. The results showed that the flowering similarity index predicts the likelihood of hybridization much better than the number of flowering-overlap days, regardless of different flowering patterns among cultivars. Thus, temporal isolation of flowering or flowering asynchrony is the most effective means in preventing hybridization between crops and their wild relatives.

  17. A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional Chinese medicinal plant, Epimedium sagittatum

    Directory of Open Access Journals (Sweden)

    Wenjun Huang


    Full Text Available Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from E. sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase and EsFLS (flavonol synthase, but not the promoters of EsDFRs (dihydroflavonol 4-reductase and EsANS (anthocyanidin synthase in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase, NtCHI (chalcone isomerase, NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived bioactive components in E

  18. In silico tools for the analysis of antibiotic biosynthetic pathways

    DEFF Research Database (Denmark)

    Weber, Tilmann


    Natural products of bacteria and fungi are the most important source for antimicrobial drug leads. For decades, such compounds were exclusively found by chemical/bioactivity-guided screening approaches. The rapid progress in sequencing technologies only recently allowed the development of novel...... screening methods based on the genome sequences of potential producing organisms. The basic principle of such genome mining approaches is to identify genes, which are involved in the biosynthesis of such molecules, and to predict the products of the identified pathways. Thus, bioinformatics methods...... and tools are crucial for genome mining. In this review, a comprehensive overview is given on programs and databases for the identification and analysis of antibiotic biosynthesis gene clusters in genomic data....

  19. Producing the Ethylene Signal: Regulation and Diversification of Ethylene Biosynthetic Enzymes. (United States)

    Booker, Matthew A; DeLong, Alison


    Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed.

  20. 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.

  1. Recent advances in Cannabis sativa research: biosynthetic studies and its potential in biotechnology. (United States)

    Sirikantaramas, Supaart; Taura, Futoshi; Morimoto, Satoshi; Shoyama, Yukihiro


    Cannabinoids, consisting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate:olivetolate geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

  2. Biosynthetic Relationship between Acutumine and Dechloroacutumine in Menispermum dauricum Root Cultures. (United States)

    Babiker, H A; Sugimoto, Y; Saisho, T; Inanaga, S; Hashimoto, M; Isogai, A


    The biosynthetic relationship between acutumine 1 and dechloroacutumine 2 was studied using (13)C-labeled tyrosine and (3)H-labeled 2 as tracers. (13)C-NMR spectra of (13)C-labeled 1 and 2 showed that the alkaloids, each composed of two molecules of tyrosine, are derived from the same biosynthetic pathway. Feeding Menispermum dauricum (Menispermaceae) roots, cultured in a chloride-enriched medium, with (3)H-labeled 2 demonstrated that 1 is the only alkaloid metabolite of 2. Conversion (5%) of the exogenously applied 2, taken up by the roots, into 1 showed that 2 is the precursor of 1. Incomplete conversion of 2 into 1 suggests accumulation of the exogenously applied 2 in cell organelles and/or compartmentation of the enzymes involved in the biosynthesis of 1.

  3. Biosynthetic Machinery Involved in Aberrant Glycosylation: Promising Targets for Development Drugs Against Cancer

    Directory of Open Access Journals (Sweden)

    Andreia eVasconcelos-dos-Santos


    Full Text Available Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. The hexosamine biosynthetic pathway (HBP is a branch of glucose metabolism that produces UDP-GlcNAc, and its derivatives, UDP-GalNAc and CMP-Neu5Ac, donor substrates used in the production of glycoproteins and glycolipids. Growing evidence demonstrates that alteration of the pool of activated substrates might lead to different glycosylation and cell signaling. It is already well established that aberrant glycosylation can modulate tumor growth and malignant transformation in different cancer types. Therefore, biosynthetic machinery involved in the assembly of aberrant glycans are becoming prominent targets for anti-tumor drugs. This review describes three classes of glycosylation, O-GlcNAcylation, N-linked and mucin type O-linked glycosylation, involved in tumor progression, their biosynthesis and highlights the available inhibitors as potential anti-tumor drugs.

  4. Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative. (United States)

    Bleeker, Petra M; Mirabella, Rossana; Diergaarde, Paul J; VanDoorn, Arjen; Tissier, Alain; Kant, Merijn R; Prins, Marcel; de Vos, Martin; Haring, Michel A; Schuurink, Robert C


    Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.

  5. Simple and efficient method for isolating cDNA fragments of lea3 genes with potential for wide application in the grasses (Poaceae). (United States)

    Yu, L; Wu, X; Tang, X; Yan, B


    cDNA fragments of lea3 genes with a high GC content (from 68 to 77%) were found in several Poaceae, including Sorghum vulgare, Saccharum officinarum, Oryza officinalis, Oryza meyeriana, Ampelocalamus calcareus, Cynodon dactylon, and Zizania latifoli. They were successfully isolated by means of optimal experimental parameters, which included dimethyl sulfoxide as additive and degenerate primers "AGETKAS" and "AGKDKTG", and their sequences were analyzed. Compared to the method of isolating genes by screening of a cDNA library using abscisic acid- and other stress-responsive cDNA clones, which is time-consuming and costly, this method is relatively easy and inexpensive. Using this new method, many new homologue lea3 genes were rapidly determined.

  6. In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method. (United States)

    Araújo, Crystiane Rodrigues; Miranda, Karla Carvalho; Fernandes, Orionalda de Fatima Lisboa; Soares, Ailton José; Silva, Maria do Rosário Rodrigues


    The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 masculineC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 microg/mL were found for 33.3%, 31.6% and 15% of isolates for itraconazole, ketoconazole and terbinafine, respectively.

  7. System and method for filling a plurality of isolated vehicle fluid circuits through a common fluid fill port (United States)

    Sullivan, Scott C; Fansler, Douglas


    A vehicle having multiple isolated fluid circuits configured to be filled through a common fill port includes a first fluid circuit disposed within the vehicle, the first fluid circuit having a first fill port, a second fluid circuit disposed within the vehicle, and a conduit defining a fluid passageway between the first fluid circuit and second fluid circuit, the conduit including a valve. The valve is configured such that the first and second fluid circuits are fluidly coupled via the passageway when the valve is open, and are fluidly isolated when the valve is closed.

  8. 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


    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

  9. Dothistroma pini, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes



    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...

  10. Diversity in biosynthetic pathways of galactolipids in the light of endosymbiotic origin of chloroplasts

    Directory of Open Access Journals (Sweden)

    Naoki eSato


    Full Text Available Cyanobacteria and chloroplasts perform oxygenic photosynthesis, and share a common origin. Galactolipids are present in the photosynthetic membranes of both cyanobacteria and chloroplasts, but the biosynthetic pathways of the galactolipids are significantly different in the two systems. In this minireview, we explain the history of the discovery of the cyanobacterial pathway, and present a probable scenario of the evolution of the two pathways.

  11. Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

    DEFF Research Database (Denmark)

    Maric, Selma; Thygesen, Mikkel Boas; Schiller, Jürgen;


    Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applicati...... tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis....

  12. Structure of Nampt/PBEF/visfatin, a mammalian NAD[superscript +]biosynthetic enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tao; Zhang, Xiangbin; Bheda, Poonam; Revollo, Javier R.; Imai, Shin-ichiro; Wolberger, Cynthia (JHU-MED); (WU-MED)


    Nicotinamide phosphoribosyltransferase (Nampt) synthesizes nicotinamide mononucleotide (NMN) from nicotinamide in a mammalian NAD{sup +} biosynthetic pathway and is required for SirT1 activity in vivo. Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of NMN shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism.


    Directory of Open Access Journals (Sweden)

    Dan Cascaval


    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.

  14. Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c

    Directory of Open Access Journals (Sweden)

    José J. Fernández


    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.

  15. Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster. (United States)

    Balakrishnan, Bijinu; Karki, Suman; Chiu, Shih-Hau; Kim, Hyun-Ju; Suh, Jae-Won; Nam, Bora; Yoon, Yeo-Min; Chen, Chien-Chi; Kwon, Hyung-Jin


    Monascus spp. produce several well-known polyketides such as monacolin K, citrinin, and azaphilone pigments. In this study, the azaphilone pigment biosynthetic gene cluster was identified through T-DNA random mutagenesis in Monascus purpureus. The albino mutant W13 bears a T-DNA insertion upstream of a transcriptional regulator gene (mppR1). The transcription of mppR1 and the nearby polyketide synthase gene (MpPKS5) was significantly repressed in the W13 mutant. Targeted inactivation of MpPKS5 also gave rise to an albino mutant, confirming that mppR1 and MpPKS5 belong to an azaphilone pigment biosynthetic gene cluster. This M. purpureus sequence was used to identify the whole biosynthetic gene cluster in the Monascus pilosus genome. MpPKS5 contains SAT/KS/AT/PT/ACP/MT/R domains, and this domain organization is preserved in other azaphilone polyketide synthases. This biosynthetic gene cluster also encodes fatty acid synthase (FAS), which is predicted to assist the synthesis of 3-oxooactanoyl-CoA and 3-oxodecanoyl-CoA. These 3-oxoacyl compounds are proposed to be incorporated into the azaphilone backbone to complete the pigment biosynthesis. A monooxygenase gene (an azaH and tropB homolog) that is located far downstream of the FAS gene is proposed to be involved in pyrone ring formation. A homology search on other fungal genome sequences suggests that this azaphilone pigment gene cluster also exists in the Penicillium marneffei and Talaromyces stipitatus genomes.

  16. Detection of vancomycin resistance in enterococcus species isolated from clinical samples and feces of colonized patients by phenotypic and genotypic methods

    Directory of Open Access Journals (Sweden)

    Priyanka Paul Biswas


    Full Text Available Background: The aim of this study was to find out the clinical correlation between the presence of vancomycin-resistant genes (van A and van B and their expression as detected by phenotypic tests in colonized patients and in clinical isolates. Materials and Methods: Enterococci were isolated from various clinical samples and also from fecal specimens of colonized patients at the time of admission, after 48 h and after 5 days of admission. Identification to species level was done using standard methods. Vancomycin susceptibility in Enterococci was detected by disc diffusion test. Minimum inhibitory concentration was determined by agar dilution method. Multiplex polymerase chain reaction (PCR was used to detect the presence of van genes. Results: Out of all the clinical and fecal samples processed, 12.0% isolates were either vancomycin resistant or vancomycin intermediate. Further, these isolates carried van A or van B genes as confirmed by PCR methods. Expression of van A gene was found to be more in Enterococcus faecalis (28.3% as compared to Enterococcus faecium (25.0% in both clinical and fecal isolates. 16.6% strains of E. faecium and 15.0% strains each of E. faecalis and Enterococcus gallinarum were found to carry van B genes. The overall prevalence of vancomycin resistant Enterococci (VRE in colonized patients was about 9.6%. Prior administration of antibiotics had significant effect (P = 0.001 on VRE carriage. Urinary tract infection was the most common infection caused by vancomycin susceptible Enterococci (VSE, 105/214 (49.0% and VRE, 13/36 (36.1%. There was no significant difference (P = 0.112 in the distribution of VRE and VSE in different infection types. Both clinical and fecal VRE showed maximum resistance to penicillin, ampicillin, and piperacillin. Resistance to linezolid was 2.8% in clinically isolated VRE. Conclusion: VRE in our study were found to be resistant to a number of commonly used antibiotics. The frequency of isolation

  17. 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)


    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

  18. 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


    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

  19. Enhancement of cordyceps polysaccharide production via biosynthetic pathway analysis in Hirsutella sinensis. (United States)

    Lin, Shan; Liu, Zhi-Qiang; Baker, Peter James; Yi, Ming; Wu, Hui; Xu, Feng; Teng, Yi; Zheng, Yu-Guo


    The addition of various sulfates for enhanced cordyceps polysaccharide (CP) production in submerged cultivation of H. sinensis was investigated, and manganese sulfate was found the most effective. 2mM of manganese sulfate on 0day (d) was investigated as the optimal adding condition, and the CP production reached optimum with 5.33%, increasing by 93.3% compared with the control. Furthermore, the consumption of three main precursors of CP was studied over cultivation under two conditions. Intracellular mannose content decreased by 43.1% throughout 6days cultivation, which corresponded to CP accumulation rate sharply increased from 0 d to 6 d, and mannose was considered as the most preferred precursor for generating CP. Subsequently, mannose biosynthetic pathway was constructed and verified for the first time in H. sinensis, which constituted the important part of CP biosynthesis, and transcriptional levels of the biosynthetic genes were studied. Transcriptional level of gene cpsA was significantly up-regulated 5.35-fold and it was a key gene involved both in mannose and CP biosynthesis. This study demonstrated that manganese sulfate addition is an efficient and simple way to improve CP production. Transcriptional analysis based on biosynthetic pathway was helpful to find key genes and better understand CP biosynthesis.

  20. A simple biosynthetic pathway for large product generation from small substrate amounts (United States)

    Djordjevic, Marko; Djordjevic, Magdalena


    A recently emerging discipline of synthetic biology has the aim of constructing new biosynthetic pathways with useful biological functions. A major application of these pathways is generating a large amount of the desired product. However, toxicity due to the possible presence of toxic precursors is one of the main problems for such production. We consider here the problem of generating a large amount of product from a potentially toxic substrate. To address this, we propose a simple biosynthetic pathway, which can be induced in order to produce a large number of the product molecules, by keeping the substrate amount at low levels. Surprisingly, we show that the large product generation crucially depends on fast non-specific degradation of the substrate molecules. We derive an optimal induction strategy, which allows as much as three orders of magnitude increase in the product amount through biologically realistic parameter values. We point to a recently discovered bacterial immune system (CRISPR/Cas in E. coli) as a putative example of the pathway analysed here. We also argue that the scheme proposed here can be used not only as a stand-alone pathway, but also as a strategy to produce a large amount of the desired molecules with small perturbations of endogenous biosynthetic pathways.

  1. A genomics based discovery of secondary metabolite biosynthetic gene clusters in Aspergillus ustus.

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    Borui Pi

    Full Text Available Secondary metabolites (SMs produced by Aspergillus have been extensively studied for their crucial roles in human health, medicine and industrial production. However, the resulting information is almost exclusively derived from a few model organisms, including A. nidulans and A. fumigatus, but little is known about rare pathogens. In this study, we performed a genomics based discovery of SM biosynthetic gene clusters in Aspergillus ustus, a rare human pathogen. A total of 52 gene clusters were identified in the draft genome of A. ustus 3.3904, such as the sterigmatocystin biosynthesis pathway that was commonly found in Aspergillus species. In addition, several SM biosynthetic gene clusters were firstly identified in Aspergillus that were possibly acquired by horizontal gene transfer, including the vrt cluster that is responsible for viridicatumtoxin production. Comparative genomics revealed that A. ustus shared the largest number of SM biosynthetic gene clusters with A. nidulans, but much fewer with other Aspergilli like A. niger and A. oryzae. These findings would help to understand the diversity and evolution of SM biosynthesis pathways in genus Aspergillus, and we hope they will also promote the development of fungal identification methodology in clinic.

  2. Comparison of microdilution and disc diffusion methods in assessing the in vitro activity of fluconazole and Melaleuca alternifolia (tea tree) oil against vaginal Candida isolates. (United States)

    Ergin, A; Arikan, S


    The in vitro activity of fluconazole and Melaleuca alternifolia (tea tree) oil was evaluated against 99 vaginal Candida strains by the broth microdilution and disc diffusion methods. The microdilution method was performed in accordance with NCCLS-M27A guidelines. An investigational method was used for the disc diffusion test. Fluconazole and tea tree oil minimum inhibitory concentrations (MICs) obtained at 48 h tended to increase 1- to 2-fold or remain the same compared to 24 h readings for most of the isolates tested. C. krusei and C. norvegensis had significantly higher MICs and smaller inhibition zones for fluconazole compared to other species. Tea tree oil MICs were found to be similar, in general, for all Candida spp. tested. The geometric mean MIC of tea tree oil for all isolates was 2.2% (range, 0.25-4%) at 24 h and 3.0% (range, 1-8%) at 48 h. Tea tree oil mean inhibition zone diameter was 24 mm (range, 14-42 mm) at 24 h and 15.8 mm (range, 10-35 mm) at 48 h. In vitro activity of tea tree oil against fluconazole-resistant Candida strains was of particular interest. The isolates had similar tea tree oil MICs and inhibition zone diameters regardless of their fluconazole susceptibility profile. Tea tree oil MIC ranges (inhibition zone diameter ranges) were 2-4% (12-21 mm) and 2% (35 mm) at 48 h for C. krusei and C. norvegensis, respectively. These results suggest that tea tree oil MICs of the fluconazole-resistant isolates are comparable to those of fluconazole-susceptible isolates. This in vitro finding is promising for potential use of topical tea tree oil formulations in the treatment of candidiasis due to fluconazole-resistant strains.

  3. Inference of Gene Flow in the Process of Speciation: An Efficient Maximum-Likelihood Method for the Isolation-with-Initial-Migration Model (United States)

    Costa, Rui J.; Wilkinson-Herbots, Hilde


    The isolation-with-migration (IM) model is commonly used to make inferences about gene flow during speciation, using polymorphism data. However, it has been reported that the parameter estimates obtained by fitting the IM model are very sensitive to the model’s assumptions—including the assumption of constant gene flow until the present. This article is concerned with the isolation-with-initial-migration (IIM) model, which drops precisely this assumption. In the IIM model, one ancestral population divides into two descendant subpopulations, between which there is an initial period of gene flow and a subsequent period of isolation. We derive a very fast method of fitting an extended version of the IIM model, which also allows for asymmetric gene flow and unequal population sizes. This is a maximum-likelihood method, applicable to data on the number of segregating sites between pairs of DNA sequences from a large number of independent loci. In addition to obtaining parameter estimates, our method can also be used, by means of likelihood-ratio tests, to distinguish between alternative models representing the following divergence scenarios: (a) divergence with potentially asymmetric gene flow until the present, (b) divergence with potentially asymmetric gene flow until some point in the past and in isolation since then, and (c) divergence in complete isolation. We illustrate the procedure on pairs of Drosophila sequences from ∼30,000 loci. The computing time needed to fit the most complex version of the model to this data set is only a couple of minutes. The R code to fit the IIM model can be found in the supplementary files of this article. PMID:28193727

  4. Identification and analysis of the paulomycin biosynthetic gene cluster and titer improvement of the paulomycins in Streptomyces paulus NRRL 8115.

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    Jine Li

    Full Text Available The paulomycins are a group of glycosylated compounds featuring a unique paulic acid moiety. To locate their biosynthetic gene clusters, the genomes of two paulomycin producers, Streptomyces paulus NRRL 8115 and Streptomyces sp. YN86, were sequenced. The paulomycin biosynthetic gene clusters were defined by comparative analyses of the two genomes together with the genome of the third paulomycin producer Streptomyces albus J1074. Subsequently, the identity of the paulomycin biosynthetic gene cluster was confirmed by inactivation of two genes involved in biosynthesis of the paulomycose branched chain (pau11 and the ring A moiety (pau18 in Streptomyces paulus NRRL 8115. After determining the gene cluster boundaries, a convergent biosynthetic model was proposed for paulomycin based on the deduced functions of the pau genes. Finally, a paulomycin high-producing strain was constructed by expressing an activator-encoding gene (pau13 in S. paulus, setting the stage for future investigations.

  5. "Paraffin wax-overlay of pour plate", a method for the isolation and enumeration of purple non-sulfur bacteria. (United States)

    Archana, A; Sasikala, Ch; Ramana, Ch V; Arunasri, K


    A modification of pour plate technique with an overlay of wax was used for isolation and enumeration of purple non-sulfur bacteria (PNSB) with equal efficiency as that of agar shake culture. The total count of PNSB ranged from 10(5)-10(8) CFU g dry soil(-1) and belonged to the genera of Rhodobacter, Rhodopseudomonas, Rhodocista and Rubrivivax.

  6. 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;


    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...

  7. Selection for phase variation of LOS biosynthetic genes frequently occurs in progression of non-typeable Haemophilus influenzae infection from the nasopharynx to the middle ear of human patients.

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    Kate L Fox

    Full Text Available Surface structures in Haemophilus influenzae are subject to rapid ON/OFF switching of expression, a process termed phase variation. We analyse tetranucleotide repeats controlling phase variation in lipo-oligosaccharide (LOS genes of H. influenzae in paired isolates from both the nasopharynx and middle ears of paediatric patients with chronic or recurrent otitis media. A change in expression of at least one of the seven phase variable LOS biosynthesis genes was seen in 12 of the 21 strain pairs. Several strains showed switching of expression in multiple LOS genes, consistent with a key role for phase variable LOS biosynthetic genes in human infection.

  8. Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. (United States)

    Alvarez, M Lucrecia; Khosroheidari, Mahdieh; Kanchi Ravi, Rupesh; DiStefano, Johanna K


    Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine.

  9. Draft Genome Sequence of Marine Sponge Symbiont Pseudoalteromonas luteoviolacea IPB1, Isolated from Hilo, Hawaii. (United States)

    Sakai-Kawada, Francis E; Yakym, Christopher J; Helmkampf, Martin; Hagiwara, Kehau; Ip, Courtney G; Antonio, Brandi J; Armstrong, Ellie; Ulloa, Wesley J; Awaya, Jonathan D


    We report here the 6.0-Mb draft genome assembly of Pseudoalteromonas luteoviolacea strain IPB1 that was isolated from the Hawaiian marine sponge Iotrochota protea Genome mining complemented with bioassay studies will elucidate secondary metabolite biosynthetic pathways and will help explain the ecological interaction between host sponge and microorganism.

  10. Draft Genome Sequence of Marine Sponge Symbiont Pseudoalteromonas luteoviolacea IPB1, Isolated from Hilo, Hawaii (United States)

    Yakym, Christopher J.; Helmkampf, Martin; Hagiwara, Kehau; Ip, Courtney G.; Antonio, Brandi J.; Armstrong, Ellie; Ulloa, Wesley J.; Awaya, Jonathan D.


    We report here the 6.0-Mb draft genome assembly of Pseudoalteromonas luteoviolacea strain IPB1 that was isolated from the Hawaiian marine sponge Iotrochota protea. Genome mining complemented with bioassay studies will elucidate secondary metabolite biosynthetic pathways and will help explain the ecological interaction between host sponge and microorganism. PMID:27660784

  11. Draft Genome Sequence of Phytopathogenic Fungus Fusarium fujikuroi CF-295141, Isolated from Pinus sylvestris (United States)

    Bertoni-Mann, Michele; Sánchez-Hidalgo, Marina; González-Menéndez, Victor


    Here, we report the draft genome sequence of a new strain of Fusarium fujikuroi, isolated from Pinus sylvestris, which was also found to produce the mycotoxin beauvericin. The Illumina-based sequence analysis revealed an approximate genome size of 44.2 Mbp, containing 164 secondary metabolite biosynthetic clusters. PMID:27795279

  12. Characterization of multi-drug resistant ESBL producing nonfermenter bacteria isolated from patients blood samples using phenotypic methods in Shiraz (Iran

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    Maneli Amin Shahidi


    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.


    Institute of Scientific and Technical Information of China (English)



    The soybean protein isolates is the soluble protein from soybeanmeal.The traditional extration method is base extraction and acid precipitation.The other methods are membrane seaperation ,foaming seaperation and biopolar electroacidification.The modification methods are thermotreat ,acid treat and enzyme treat.%概述了大豆分离蛋白的传统提取法、膜分离法、泡沫分离法、双极膜法及其通常所用的改性方法:热处理法,酸处理法和酶处理法。

  14. Comparative SNP diversity among four Eucalyptus species for genes from secondary metabolite biosynthetic pathways

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    Foley William J


    Full Text Available Abstract Background There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in Eucalyptus. With this approach the occurrence and patterns of SNP variation for a set of genes can be compared across different species from the same genus. Results In a single GS-FLX run, we sequenced over 103 Mbp and assembled them to approximately 50 kbp of reference sequences. An average sequencing depth of 315 reads per nucleotide site was achieved for all four eucalypt species, Eucalyptus globulus, E. nitens, E. camaldulensis and E. loxophleba. We sequenced 23 genes from 1,764 individuals and discovered 8,631 SNPs across the species, with about 1.5 times as many SNPs per kbp in the introns compared to exons. The exons of the two closely related species (E. globulus and E. nitens had similar numbers of SNPs at synonymous and non-synonymous sites. These species also had similar levels of SNP diversity, whereas E. camaldulensis and E. loxophleba had much higher SNP diversity. Neither the pathway nor the position in the pathway influenced gene diversity. The four species share between 20 and 43% of the SNPs in these genes. Conclusion By using conservative statistical detection methods, we were confident about the validity of each SNP. With numerous individuals sampled over the geographical range of each species, we discovered one SNP in every 33 bp for E. nitens and one in every 31 bp in E. globulus. In contrast, the more distantly related species contained more SNPs: one in every 16 bp for E. camaldulensis and one in 17 bp for E. loxophleba, which is, to the best of our knowledge, the highest frequency of SNPs

  15. Biosynthetic origin of geosmin in red beets (Beta vulgaris L.). (United States)

    Lu, Guiping; Edwards, Charles G; Fellman, John K; Mattinson, D Scott; Navazio, John


    Geosmin provides the characteristic but sometimes undesirable "earthy" flavor to red table beets. To date, it is not known whether geosmin is a byproduct of beet metabolism or synthesized by soil-borne microorganisms and taken up by the beets during maturation. Analysis of mature beet roots revealed that peels contained 6 times the amount of geosmin compared to the bodies and cores. Sterilized beet seeds were aseptically grown in a basal medium prior to analysis for the presence of geosmin. Using a headspace solid-phase microextraction (HSPME) method, the relative recovery of geosmin from beet seedling extracts was 72.0 +/- 4.2% with (-)-menthone as the internal standard. The presence of geosmin in aseptically grown beet seedlings was confirmed by gas chromatography-mass spectrometry using authentic geosmin as the standard. During aseptic growth, the concentration of geosmin in seedlings remained constant for up to 5 months but increased at 6 months. Geosmin added to the growth medium was not absorbed by the seedlings. These studies support the conclusion that red beets are capable of endogenous synthesis of geosmin.

  16. 粪便中大肠埃希菌分离方法的筛选%Screening of E. colt Isolation Methods from Feces

    Institute of Scientific and Technical Information of China (English)

    蒋翰鹏; 曹宾霞; 杨玉敏


    比较了滤膜法、涂布法和纸片法时粪便中大肠埃希菌的分离效果.通过对分离粪便大肠埃希菌的数量可知,纸片法与m-TEC培养基上滤膜法分离的大肠埃希菌数量结果基本一致.m-TEC培养基滤膜法分离的大肠埃希菌平均数量分别是伊红美蓝培养基涂布法分离大肠埃希菌平均数量的1.4倍、伊红美蓝培养基滤膜法分离大肠埃希菌平均数量的2.8倍、m-TEC培养基涂布法分离大肠埃希菌平均数量的2.25倍.分离粪便样品中大肠埃希菌选择滤膜法用m-TEC培养基进行分离为最佳分离方法.%The effects of E. colt isolation methods were compared with filtration membrane method, spreading meth-od, and paper sheet method. From the quantity of isolated E. colt from feces it was known that the results of paper sheet method were basically identical to m-TEC medium filtration membrane method~ The average quantity of E. coli isolated with m-TEC medium filtration membrane method was i. 4 times that of eosin methylene blue medium spreading method, 2.8 times that of eosin methylene blue medium filtration membrane method, 2.25 times that of m-TEC medi-um spreading method. Therefore, m-TEC medium filtration membrane method was the best method to isolate E. coli from fecal samples.

  17. Toxigenic potentiality of Aspergillus flavus and Aspergillus parasiticus strains isolated from black pepper assessed by an LC-MS/MS based multi-mycotoxin method. (United States)

    Yogendrarajah, Pratheeba; Devlieghere, Frank; Njumbe Ediage, Emmanuel; Jacxsens, Liesbeth; De Meulenaer, Bruno; De Saeger, Sarah


    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.

  18. A rapid and efficient method for isolating high quality DNA from leaves of carnivorous plants from the Drosera genus. (United States)

    Biteau, Flore; Nisse, Estelle; Hehn, Alain; Miguel, Sissi; Hannewald, Paul; Bourgaud, Frédéric


    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.

  19. Methods for axolotl blood collection, intravenous injection, and efficient leukocyte isolation from peripheral blood and the regenerating limb. (United States)

    Debuque, Ryan J; Godwin, James W


    The vertebrate immune system comprises both adaptive and innate immune cells with distinct functions during the resolution of inflammation and wound healing after tissue injury. Recent evidence implicates a requirement for innate immune cells from the myeloid lineage during the early stages of limb regeneration in the Mexican axolotl. Understanding the functions of innate and adaptive immune cells in the axolotl has been hampered by a lack of approaches to isolate and analyze these cells. Here we describe a protocol to isolate myeloid cells from the regenerating axolotl limb that incorporates intravenous delivery of physiological labels. In addition we provide a protocol to enrich for leukocytes in the peripheral blood. These protocols produce single-cell suspensions that can be analyzed using flow cytometry or sorted into specific subsets using fluorescent-activated cell sorting (FACS). FACS is a routine approach to sort cells based on their physical characteristics as well as their cell surface antigen repertoire. Isolated cell populations can then be analyzed in a wide range of downstream assays to facilitate a greater understanding of leukocyte biology in the axolotl.


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    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.