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Sample records for agarose

  1. Oxygen-17 relaxation in aqueous agarose gels

    International Nuclear Information System (INIS)

    Ablett, S.; Lillford, P.J.

    1977-01-01

    Nuclear magnetic relaxation of oxygen-17 in H 2 17 O enriched agarose gels shows that existing explanations of water behaviour are oversimplified. Satisfactory models must include at least three proton phases, two of which involve water molecules. (Auth.)

  2. Crosslinking of agarose bioplastic using citric acid.

    Science.gov (United States)

    Awadhiya, Ankur; Kumar, David; Verma, Vivek

    2016-10-20

    We report chemical crosslinking of agarose bioplastic using citric acid. Crosslinking was confirmed using Fourier transform infrared (FTIR) spectroscopy. The effects of crosslinking on the tensile strength, swelling, thermal stability, and degradability of the bioplastic were studied in detail. The tensile strength of the bioplastic films increased from 25.1MPa for control films up to a maximum of 52.7MPa for citric acid crosslinked films. At 37°C, the amount of water absorbed by crosslinked agarose bioplastic was only 11.5% of the amount absorbed by non-crosslinked controls. Thermogravimetric results showed that the crosslinked samples retain greater mass at high temperature (>450°C) than control samples. Moreover, while the crosslinked films were completely degradable, the rate of degradation was lower compared to non-crosslinked controls. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Composition of agarose substrate affects behavioral output of Drosophila larvae

    Directory of Open Access Journals (Sweden)

    Anthi Aristomenis Apostolopoulou

    2014-01-01

    Full Text Available In the last decade the Drosophila larva has evolved into a simple model organism offering the opportunity to integrate molecular genetics with systems neuroscience. This led to a detailed understanding of the functional neuronal networks for a number of sensory functions and behaviors including olfaction, vision, gustation and learning and memory. Typically, behavioral assays in use exploit simple Petri dish setups with either agarose or agar as a substrate. However, neither the quality nor the concentration of the substrate is generally standardized across these experiments and there is no data available on how larval behavior is affected by such different substrates. Here, we have investigated the effects of different agarose concentrations on several larval behaviors. We demonstrate that agarose concentration is an important parameter, which affects all behaviors tested: preference, feeding, learning and locomotion. Larvae can discriminate between different agarose concentrations, they feed differently on them, they can learn to associate an agarose concentration with an odor stimulus and crawl faster on a substrate of higher agarose concentration. Additionally, we have investigated the effect of agarose concentration on three quinine based behaviors: preference, feeding and learning. We show that in all cases examined the behavioral output changes in an agarose concentration-dependent manner. Our results suggest that comparisons between experiments performed on substrates differing in agarose concentration should be done with caution. It should be taken into consideration that the agarose concentration can affect the behavioral output and thereby the experimental outcomes per se potentially due to an increased escape response on more rigid substrates.

  4. Agarose gel electrophoresis for the separation of DNA fragments.

    Science.gov (United States)

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-04-20

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate

  5. Functionalized Agarose Self-Healing Ionogels Suitable for Supercapacitors.

    Science.gov (United States)

    Trivedi, Tushar J; Bhattacharjya, Dhrubajyoti; Yu, Jong-Sung; Kumar, Arvind

    2015-10-12

    Agarose has been functionalized (acetylated/carbanilated) in an ionic liquid (IL) medium of 1-butyl-3-methylimidazolium acetate at ambient conditions. The acetylated agarose showed a highly hydrophobic nature, whereas the carbanilated agarose could be dissolved in water as well as in the IL medium. Thermoreversible ionogels were obtained by cooling the IL sols of carbanilated agarose at room temperature. The ionogel prepared from a protic-aprotic mixed-IL system (1-butyl-3-methylimidazolium chloride and N-(2-hydroxyethyl)ammonium formate) demonstrated a superior self-healing property, as confirmed from rheological measurements. The superior self-healing property of such an ionogel has been attributed to the unique inter-intra hydrogen-bonding network of functional groups inserted in the agarose. The ionogel was tested as a flexible solid electrolyte for an activated-carbon-based supercapacitor cell. The measured specific capacitance was found to be comparable with that of a liquid electrolyte system at room temperature and was maintained for up to 1000 charge-discharge cycles. Such novel functionalized-biopolymer self-healing ionogels with flexibility and good conductivity are desirable for energy-storage devices and electronic skins with superior lifespans and robustness. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Preparation and Characterization of Chitosan—Agarose Composite Films

    Directory of Open Access Journals (Sweden)

    Zhang Hu

    2016-09-01

    Full Text Available Nowadays, there is a growing interest to develop biodegradable functional composite materials for food packaging and biomedicine applications from renewable sources. Some composite films were prepared by the casting method using chitosan (CS and agarose (AG in different mass ratios. The composite films were analyzed for physical-chemical-mechanical properties including tensile strength (TS, elongation-at-break (EB, water vapor transmission rate (WVTR, swelling ratio, Fourier-transform infrared spectroscopy, and morphology observations. The antibacterial properties of the composite films were also evaluated. The obtained results reveal that an addition of AG in varied proportions to a CS solution leads to an enhancement of the composite film’s tensile strength, elongation-at-break, and water vapor transmission rate. The composite film with an agarose mass concentration of 60% was of the highest water uptake capacity. These improvements can be explained by the chemical structures of the new composite films, which contain hydrogen bonding interactions between the chitosan and agarose as shown by Fourier-transform infrared spectroscopy (FTIR analysis and the micro-pore structures as observed with optical microscopes and scanning electron microscopy (SEM. The antibacterial results demonstrated that the films with agarose mass concentrations ranging from 0% to 60% possessed antibacterial properties. These results indicate that these composite films, especially the composite film with an agarose mass concentration of 60%, exhibit excellent potential to be used in food packaging and biomedical materials.

  7. In vivo biocompatibility evaluation of Cibacron blue-agarose.

    Science.gov (United States)

    Kao, J M; Rose, R; Yousef, M; Hunter, S K; Rodgers, V G

    1999-12-15

    This study investigated the biocompatibility of Cibacron blue-agarose as a biomaterial for microencapsulation. Cibacron blue-agarose is known to have an affinity for albumin under certain pH conditions and in the proper steric environment. Thus it was postulated that the material's high affinity for host albumin might reduce a secondary immune response and reduce the fibrotic overgrowth that often accompanies transplanted foreign materials. In vivo tests were performed using the Lewis rat model. Both Cibacron blue-agarose and plain agarose disks were prepared, with some disks from each group being pre-exposed to sera from Lewis rats. The disks were transplanted into the peritoneal cavities of Lewis rats. After 115 days the disks were excised. Fibrotic overgrowth was analyzed using light microscopy, and a blind study was used to measure the average growth thickness on each disk. The results demonstrated that all disks developed some fibrotic encapsulation and that the presence of Cibacron blue was not significant in reducing fibrotic overgrowth (p = 0.62). Agarose disks pre-exposed to sera had significantly less average overgrowth than any other group (p = 0. 06). Copyright 1999 John Wiley & Sons, Inc.

  8. Textural Properties of Agarose Gels described by FT-Rheology

    NARCIS (Netherlands)

    Klein, C.O.; Venema, P.; Sagis, L.M.C.; Linden, van der E.

    2008-01-01

    Large Amplitude Oscillatory Shear was used to determine the non-linear rheological properties of agarose gels. The analysis was performed with the characteristic functions method based on FT-Rheology, that gives access to a physical interpretation of the non-linear regime. This analysis was then

  9. Blood grouping based on PCR methods and agarose gel electrophoresis.

    Science.gov (United States)

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  10. Rapid transfer of DNA from agarose gels to nylon membranes.

    OpenAIRE

    Reed, K C; Mann, D A

    1985-01-01

    The unique properties of nylon membranes allow for dramatic improvement in the capillary transfer of DNA restriction fragments from agarose gels (Southern blotting). By using 0.4 M NaOH as the transfer solvent following a short pre-treatment of the gel in acid, DNA is depurinated during transfer. Fragments of all sizes are eluted and retained quantitatively by the membrane; furthermore, the alkaline solvent induces covalent fixation of DNA to the membrane. The saving in time and materials aff...

  11. Thermoresponsive chitosan-agarose hydrogel for skin regeneration.

    Science.gov (United States)

    Miguel, Sónia P; Ribeiro, Maximiano P; Brancal, Hugo; Coutinho, Paula; Correia, Ilídio J

    2014-10-13

    Healing enhancement and pain control are critical issues on wound management. So far, different wound dressings have been developed. Among them, hydrogels are the most applied. Herein, a thermoresponsive hydrogel was produced using chitosan (deacetylation degree 95%) and agarose. Hydrogel bactericidal activity, biocompatibility, morphology, porosity and wettability were characterized by confocal microscopy, MTS assay and SEM. The performance of the hydrogel in the wound healing process was evaluated through in vivo assays, during 21 days. The attained results revealed that hydrogel has a pore size (90-400 μm) compatible with cellular internalization and proliferation. A bactericidal activity was observed for hydrogels containing more than 188 μg/mL of chitosan. The improved healing and the lack of a reactive or a granulomatous inflammatory reaction in skin lesions treated with hydrogel demonstrate its suitability to be used in a near future as a wound dressing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

    Directory of Open Access Journals (Sweden)

    Song-I Chun

    Full Text Available A reference reagent, 3-(trimethylsilyl propionic-2, 2, 3, 3-d4 acid sodium (TSP, has been used frequently in nuclear magnetic resonance (NMR and magnetic resonance spectroscopy (MRS as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells.A human osteosarcoma cell line (MG-63 was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding.In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation and cell viability. High concentrations of TSP (from 10 to 30 mM reduced both cell proliferation and viability (to 30% of the control after one week of exposure, but no such effects were found using low concentrations of TSP (0-10 mM.This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

  13. High-quality substrate for fluorescence enhancement using agarose-coated silica opal film.

    Science.gov (United States)

    Xu, Ming; Li, Juan; Sun, Liguo; Zhao, Yuanjin; Xie, Zhuoying; Lv, Linli; Zhao, Xiangwei; Xiao, Pengfeng; Hu, Jing; Lv, Mei; Gu, Zhongze

    2010-08-01

    To improve the sensitivity of fluorescence detection in biochip, a new kind of substrates was developed by agarose coating on silica opal film. In this study, silica opal film was fabricated on glass substrate using the vertical deposition technique. It can provide stronger fluorescence signals and thus improve the detection sensitivity. After coating with agarose, the hybrid film could provide a 3D support for immobilizing sample. Comparing with agarose-coated glass substrate, the agarose-coated opal substrates could selectively enhance particular fluorescence signals with high sensitivity when the stop band of the silica opal film in the agarose-coated opal substrate overlapped the fluorescence emission wavelength. A DNA hybridization experiment demonstrated that fluorescence intensity of special type of agarose-coated opal substrates was about four times that of agarose-coated glass substrate. These results indicate that the optimized agarose-coated opal substrate can be used for improving the sensitivity of fluorescence detection with high quality and selectivity.

  14. Recovery of DNA from agarose gel by trap method | Xia | African ...

    African Journals Online (AJOL)

    Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be ...

  15. Preparation of gold nanoparticles-agarose gel composite and its application in SERS detection

    Science.gov (United States)

    Ma, Xiaoyuan; Xia, Yu; Ni, Lili; Song, Liangjing; Wang, Zhouping

    2014-03-01

    Agarose gel/gold nanoparticles hybrid was prepared by adding gold nanoparticles to preformed agarose gel. Nanocomposite structures and properties were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and UV-Vis-NIR absorption spectroscopy. Based on the swelling-contraction characteristics of agarose gel and the adjustable localized surface plasmon resonance (LSPR) of the gold nanoparticles, the nanocomposites were used as surface enhanced Raman scattering (SERS) substrate to detect the Raman signal molecules (NBA, MBA, 1NAT). Results revealed that the porous structure of the agarose gel provided a good carrier for the enrichment of the gold nanoparticles. The gold nanoparticles dynamic hot-spot effect arising from the agarose gel contraction loss of water in the air greatly enhanced the Raman signal. Furthermore, the gel could be cleaned with washing solution and recycling could be achieved for Raman detection.

  16. Laminin active peptide/agarose matrices as multifunctional biomaterials for tissue engineering.

    Science.gov (United States)

    Yamada, Yuji; Hozumi, Kentaro; Aso, Akihiro; Hotta, Atsushi; Toma, Kazunori; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

    2012-06-01

    Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Proteoglycon synthesis by articular chondrocytes in agarose culture

    International Nuclear Information System (INIS)

    Sweet, M.B.E.; Grisillo, A.; Coehlo, A.; Schnitzler, C.M.

    1987-01-01

    Articular chondrocytes were isolated from knee joints of full-term bovine foetuses and grown in long-term agarose cultures. At intervals, cultures were labelled with 35 S-[sulphate] or D[6- 3 H] glucosamine. Newly synthesized proteoglycans were extracted with 4 M guanidine HCl and purified by isopycnic density gradient centrifugation or on DEAE cellulose in the presence of 8 M urea. Characterization of the proteoglycans revealed them to be identical in size to those present in the tissue and to be similarly capable of aggregation with hyaluronate. Newly synthesized chondroitin sulphate chains were identical in size, but newly synthesized keratan sulphate chains were somewhat larger than those present in the tissue. The newly synthesized proteoglycans were shown to contain the same range of O-linked oligosaccharides identified in proteoglycans of the Swarm rat chondrosarcoma. Cartilage-specific proteoglycan continued to be synthesized by the chondrocytes for up to 60 days; however, with time, proportionately more of a small non-aggregating proteoglycan appeared

  18. Films of Agarose Enable Rapid Formation of Giant Liposomes in Solutions of Physiologic Ionic Strength

    OpenAIRE

    Horger, Kim S.; Estes, Daniel J.; Capone, Ricardo; Mayer, Michael

    2009-01-01

    This paper describes a method to form giant liposomes in solutions of physiologic ionic strength, such as phosphate buffered saline (PBS) or 150 mM KCl. Formation of these cell-sized liposomes proceeded from hybrid films of partially dried agarose and lipids. Hydrating the films of agarose and lipids in aqueous salt solutions resulted in swelling and partial dissolution of the hybrid films and in concomitant rapid formation of giant liposomes in high yield. This method did not require the pre...

  19. Ultra-deep desulfurization via reactive adsorption on peroxophosphomolybdate/agarose hybrids.

    Science.gov (United States)

    Xu, Jian; Li, Huacheng; Wang, Shengtian; Luo, Fang; Liu, Yunyu; Wang, Xiaohong; Jiang, Zijiang

    2014-09-01

    A catalyst system composed of peroxophosphomolybdates as catalytic center and agarose as matrix material had been designed. The [C16H33N(CH3)3]3[PO4{MoO(O2)2}4]/agarose (C16PMo(O2)2/agarose) hybrid was found to be active for oxidation desulfurization (ODS) of dibenzothiophene (DBT) or real fuel into corresponding sulfone by H2O2 as an oxidant, while the sulfur content could be reduced to 5ppm. The higher activity comes from its components including [PO4{MoO(O2)2}4] catalytic sites, the hydrophobic quaternary ammonium cation affinity to low polarity substrates, and agarose matrix affinity to H2O2 and sulfone. During the oxidative reaction, the mass transfer resistance between H2O2 and organic sulfurs could be decreased and the reaction rate could increase by the assistance of agarose and hydrophobic tails of [C16H33N(CH3)3]3[PO4{MoO(O2)2}4]. Meanwhile, the oxidative products could be adsorbed by agarose matrix to give clean fuel avoiding the post-treatment. In addition, the hybrid was easily regenerated to be reused. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    Energy Technology Data Exchange (ETDEWEB)

    Dumpala, Pradeep R., E-mail: pdumpala@rixd.org [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Parker, Thomas S.; Levine, Daniel M. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); Smith, Barry H. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); NewYork-Presbyterian Hospital, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021 (United States); Gazda, Lawrence S. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States)

    2016-08-05

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  1. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    International Nuclear Information System (INIS)

    Dumpala, Pradeep R.; Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A.; Parker, Thomas S.; Levine, Daniel M.; Smith, Barry H.; Gazda, Lawrence S.

    2016-01-01

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  2. In situ observation of sol-gel transition of agarose aqueous solution by fluorescence measurement.

    Science.gov (United States)

    Wang, Zheng; Yang, Kun; Li, Haining; Yuan, Chaosheng; Zhu, Xiang; Huang, Haijun; Wang, Yongqiang; Su, Lei; Fang, Yapeng

    2018-06-01

    Sol-gel transition behavior of agarose aqueous solution was investigated by using rheology and fluorescence measurement. On heating, the storage modulus G' decreased gradually, then deviated abruptly at the temperature of about 65°C, and finally decreased slowly again. For fluorescence measurement, the phase transition point kept almost at the temperature of 65°C, which was consistent with that in rheology measurement. Upon compression, it was indicated that the fluorescence lifetime for the probe in the agarose aqueous solution showed a dramatic change in the vicinity of the phase transition point. T vs. P phase diagram of agarose aqueous solution was constructed, which showed that the melting point was an increasing function of pressure. Based on the phase diagram, the agarose gels were prepared by cooling under atmospheric pressure and the pressure of 300MPa, respectively. From the result of the recovered samples studied by optical rheometry, it was found that agarose gel prepared under high pressure had a higher elasticity and lower viscosity index, compared with that under atmospheric pressure. It could be speculated that such kinds of properties might be attributed to the smaller pore size during gelation under high pressure. Copyright © 2018. Published by Elsevier B.V.

  3. Quantitative determination of glycine in aqueous solution using glutamate dehydrogenase-immobilized glyoxal agarose beads.

    Science.gov (United States)

    Keskin, Semra Yilmazer; Keskin, Can Serkan

    2014-01-01

    In this study, an enzymatic procedure for the determination of glycine (Gly) was developed by using a column containing immobilized glutamate dehydrogenase (GDH) on glyoxal agarose beads. Ammonia is produced from the enzymatic reactions between Gly and GDH with NAD(+) in phosphate buffer medium. The indophenol blue method was used for ammonia detection based on the spectrophotometric measurements of blue-colored product absorbing at 640 nm. The calibration graph is linear in the range of 0.1-10 mM of Gly concentrations. The effect of pH, temperature, and time interval was studied to find column stability, and also the interference effects of other amino acids was investigated. The interaction between GDH and glyoxal agarose beads was analyzed by Fourier transform infrared (FTIR) spectroscopy. The morphology of the immobilized and non-immobilized agarose beads were characterized by atomic force microscopy (AFM).

  4. Fabrication of Self-Healable and Patternable Polypyrrole/Agarose Hybrid Hydrogels for Smart Bioelectrodes.

    Science.gov (United States)

    Park, Nokyoung; Chae, Seung Chul; Kim, Il Tae; Hur, Jaehyun

    2016-02-01

    We present a new class of electrically conductive, mechanically moldable, and thermally self-healable hybrid hydrogels. The hybrid gels consist of polypyrrole and agarose as the conductive component and self-healable matrix, respectively. By using the appropriate oxidizing agent under conditions of mild temperature, the polymerization of pyrrole occurred along the three-dimensional network of the agarose hydrogel matrix. In contrast to most commercially available hydrogels, the physical crosslinking of agarose gel allows for reversible gelation in the case of our hybrid gel, which could be manipulated by temperature variation, which controls the electrical on/off behavior of the hybrid gel electrode. Exploiting this property, we fabricated a hybrid conductive hydrogel electrode which also self-heals thermally. The novel composite material we report here will be useful for many technological and biological applications, especially in reactive biomimetic functions and devices, artificial muscles, smart membranes, smart full organic batteries, and artificial chemical synapses.

  5. Enzymatic liquefaction of agarose above the sol-gel transition temperature using a thermostable endo-type β-agarase, Aga16B.

    Science.gov (United States)

    Kim, Jung Hyun; Yun, Eun Ju; Seo, Nari; Yu, Sora; Kim, Dong Hyun; Cho, Kyung Mun; An, Hyun Joo; Kim, Jae-Han; Choi, In-Geol; Kim, Kyoung Heon

    2017-02-01

    The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40 T , was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

  6. Immunoperoxidase staining and radioimmunobinding of human tumor markers separated by direct tissue agarose isoelectric focusing

    International Nuclear Information System (INIS)

    Saravis, C.A.; Cunningham, C.G.; Marasco, P.V.; Cook, R.B.; Zamcheck, N.; FMC Corp., Rockland, ME

    1980-01-01

    The new technique of agarose isoelectric focusing is used to identify, quantitate, and characterize specific tumor markers. After fixation of the isoelectric focusing patterns these are reacted with specific anti-tumor marker antisera, then with second antibody either peroxidase conjugated or radiolabellad (radioiodine). (RB) [de

  7. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

    Science.gov (United States)

    Stellwagen, Nancy C.

    2009-01-01

    This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due primarily to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 ± 0.01) × 10-4 cm2/Vs in 40 mM Tris-acetate-EDTA buffer at 20°C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration. PMID:19517510

  8. A simple immunoblotting method after separation of proteins in agarose gel

    DEFF Research Database (Denmark)

    Koch, C; Skjødt, K; Laursen, I

    1985-01-01

    A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence ...

  9. Preparation of berbamine loaded chitosan-agarose microspheres and in vitro release study

    Directory of Open Access Journals (Sweden)

    Zhang Hu

    2012-01-01

    Full Text Available Berbamine loaded chitosan-agarose microspheres were prepared using a water-in-oil emulsion technique. Optimum preparing parameters were determined by orthogonal experiments as follows: ratio of berbamine to chitosan (w/w is 1:10; percentage of emulsifier (span 80, v/v is 6%; volume of glutaraldehyde is 2 mL; and reaction temperature is 70 ºC. Under these optimal conditions, the encapsulation efficiency and loading capacity of microspheres are 84.57% and 8.44%, respectively. The swelling tests showed that the microspheres possessed higher swelling ratio at pH 7.4 than at pH 1.2. FTIR indicated that berbamine had been successfully loaded in the chitosan-agarose microspheres by physical entrapment. In vitro release studies showed that berbamine was released from microspheres in a significantly sustained fashion.

  10. Structural aspects of magnetic fluid stabilization in aqueous agarose solutions

    Energy Technology Data Exchange (ETDEWEB)

    Nagornyi, A.V. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Taras Shevchenko National University of Kyiv, Kyiv (Ukraine); Petrenko, V.I., E-mail: vip@nf.jinr.ru [Joint Institute for Nuclear Research, Dubna (Russian Federation); Taras Shevchenko National University of Kyiv, Kyiv (Ukraine); Avdeev, M.V. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Yelenich, O.V.; Solopan, S.O.; Belous, A.G. [V.I.Vernadsky Institute of General and Inorganic Chemistry of the Ukrainian NAS, Kyiv (Ukraine); Gruzinov, A.Yu. [National Research Centre “Kurchatov Institute”, Moscow (Russian Federation); Ivankov, O.I. [Joint Institute for Nuclear Research, Dubna (Russian Federation); Institute for Safety Problems of Nuclear Power Plants of the Ukrainian NAS, Kyiv (Ukraine); Bulavin, L.A. [Taras Shevchenko National University of Kyiv, Kyiv (Ukraine); Institute for Safety Problems of Nuclear Power Plants of the Ukrainian NAS, Kyiv (Ukraine)

    2017-06-01

    Structure characterization of magnetic fluids (MFs) synthesized by three different methods in aqueous solutions of agarose was done by means of small-angle neutron (SANS) and synchrotron X-ray scattering (SAXS). The differences in the complex aggregation observed in the studied magnetic fluids were related to different stabilizing procedures of the three kinds of MFs. The results of the analysis of the scattering (mean size of single polydisperse magnetic particles, fractal dimensions of the aggregates) are consistent with the data of transmission electron microscopy (TEM). - Highlights: • MFs synthesized by three different methods in agarose solution were studied. • all MFs are agglomerated colloidal systems whose structures are nevertheless stable in time. • differences in the complex aggregation were observed in the studied magnetic fluids. • results of the SAXS and SANS analysis are consistent with TEM data.

  11. Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

    DEFF Research Database (Denmark)

    Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter

    2015-01-01

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation...... structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis....... measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary...

  12. Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

    OpenAIRE

    Rosenbluh, A; Nir, R; Sahar, E; Rosenberg, E

    1989-01-01

    Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficie...

  13. Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis.

    Science.gov (United States)

    Riond, Barbara; Wenger-Riggenbach, Bettina; Hofmann-Lehmann, Regina; Lutz, Hans

    2009-03-01

    Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and alpha-, beta-, and gamma-globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm-blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi-automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within-run and within-assay precision. Data from warm-blooded and draught horses were compared using the Mann-Whitney U test. Within-run and within-assay CVs were draught horses and so combined reference intervals (2.5-97.5%) were calculated for total protein (51.0-72.0 g/L), albumin (29.6-38.5 g/L), alpha(1)-globulin (1.9-3.1 g/L), alpha(2)-globulin (5.3-8.7 g/L), beta(1)-globulin (2.8-7.3g/L), beta(2)-globulin (2.2-6.0 g/L), and gamma-globulin (5.8-12.7 g/L) concentrations, and albumin/globulin ratio (0.93-1.65). Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.

  14. High throughput generation and trapping of individual agarose microgel using microfluidic approach

    KAUST Repository

    Shi, Yang; Gao, Xinghua; Chen, Longqing; Zhang, Min; Ma, Jingyun; Zhang, Xixiang; Qin, Jianhua

    2013-01-01

    Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

  15. The Influence of Conditioning Agent on Phosphate Diffusion Coefficient through Polyacrylamide and Agarose Gel

    Directory of Open Access Journals (Sweden)

    Layta Dinira

    2013-03-01

    Full Text Available Excess phosphate in natural water can cause algae grow rapidly, to the extent causing many fish deaths that led to the extinction of certain species. Therefore, an analysis or periodic observations of phosphate levels in the water is needed. The commonly used method is diffusive gradient in thin films (DGT technique. The DGT technique is based on the ability of analyte to diffuse through a gel, which have a value named diffusion coefficient. This research was conducted in order to study the effect of different storage solution to the phosphate diffusion coefficient through polyacrylamide and agarose gels. Initial research performed with making the polyacrylamide and agarose gels. To observe the effect of different storage solutions, the gels partly stored in distilled water gel while the others are stored in a NaCl solution of 0.01 M. Phosphate diffusion coefficient was determined using Fick's Law after analyze the phosphate concentration using UV-Visible spectrophotometer. The results showed that phosphate diffusion coefficient was highest when polyacrylamide and agarose gels stored in NaCl solution of 0.01 M.

  16. Method Development for Extraction of Butyrylcholin- esterase using Protein-G Agarose Spin Columns

    Directory of Open Access Journals (Sweden)

    Amruta S. Indapurkar

    2015-01-01

    Full Text Available Butyrylcholinesterase (BuChE is a biomarker of organophosphate (OP poisoning and can be used as a diagnostic marker to measure exposure to OP compounds. The purpose of this study was to develop a method to extract BuChE from human plasma. BuChE was extracted from plasma using the NAb protein-G Agarose Spin Kit. Factors affecting extraction like incubation time, plasma volume and cross-linking of antibodies to agarose beads were evaluated. All samples were analyzed for BuChE activity using the Ellman’s assay. The incubation times of plasma and anti-BuChE antibodies marginally affected the extraction efficiency of BuChE whereas a decrease in plasma volume increased the extraction efficiency. Cross-linking of anti-BuChE antibodies on agarose increased the extraction efficiency. The NAb protein-G Spin Kit can be used successfully to extract BuChE from human plasma. This extraction technique may be coupled to downstream analytical analyses for diagnosing exposure to OP compounds.

  17. Passive detection of Pb in water using rock phosphate agarose beads.

    Science.gov (United States)

    Edenborn, Harry M; Howard, Bret H; Sams, James I; Vesper, Dorothy J; Edenborn, Sherie L

    2017-08-15

    In this study, passive detectors for Pb were prepared by immobilizing powdered rock phosphate in agarose beads. Rock phosphate has been used to treat Pb-contaminated waters and soil by fixing the metal as an insoluble pyromorphite mineral. Under lab conditions, Pb was rapidly adsorbed from aqueous solution by the beads over time, consistent with the acidic dissolution of rock phosphate, the precipitation of pyromorphite within the pore space of the agarose gel matrix, and surface exchange reactions. Net accumulation of Pb occurred when beads were exposed to simulated periodic releases of Pb over time. Under field conditions, beads in mesh bags were effective at detecting dissolved Pb being transported as surface runoff from a site highly contaminated with Pb. Rates of Pb accumulation in beads under field conditions appeared to be correlated with the frequency of storm events and total rainfall. The rock phosphate agarose bead approach could be an inexpensive way to carry out source-tracking of Pb pollution, to verify the successful remediation of sites with Pb-contaminated soil, and to routinely monitor public water systems for potential Pb contamination. Published by Elsevier B.V.

  18. High throughput generation and trapping of individual agarose microgel using microfluidic approach

    KAUST Repository

    Shi, Yang

    2013-02-28

    Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

  19. Relative Humidity Sensor Based on No-Core Fiber Coated by Agarose-Gel Film

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2017-10-01

    Full Text Available A relative humidity (RH sensor based on single-mode–no-core–single-mode fiber (SNCS structure is proposed and experimentally demonstrated. The agarose gel is coated on the no-core fiber (NCF as the cladding, and multimode interference (MMI occurs in the SNCS structure. The transmission spectrum of the sensor is modulated at different ambient relative humidities due to the tunable refractive index property of the agarose gel film. The relative humidity can be measured by the wavelength shift and intensity variation of the dip in the transmission spectra. The humidity response of the sensors, coated with different concentrations and coating numbers of the agarose solution, were experimentally investigated. The wavelength and intensity sensitivity is obtained as −149 pm/%RH and −0.075 dB/%RH in the range of 30% RH to 75% RH, respectively. The rise and fall time is tested to be 4.8 s and 7.1 s, respectively. The proposed sensor has a great potential in real-time RH monitoring.

  20. Aqueous phase catalytic conversion of agarose to 5-hydroxymethylfurfural by metal chlorides

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Lishi; Laskar, Dhrubojyoti D.; Lee, Suh-Jane; Yang, Bin

    2013-12-14

    Abstract: 5-HMF is a key intermediate for producing chemicals and fuels that can substitute for today’s petroleum-derived feedstocks. A series of metal chlorides, including NaCl, CaCl2, MgCl2, ZnCl2, CuCl2, FeCl3, and CrCl3, were comparatively investigated to catalyze agarose degradation for production of 5-HMF at temperature 180 oC, 200 oC, and 220 oC for 30 min, with catalyst concentration of 0.5% (w/w), 1% (w/w) and 5% (w/w), and substrate concentration of 2% (w/w). Our results revealed that alkali metal chlorides and alkali earth metal chlorides such as NaCl, CaCl2 and MgCl2 gave better 5-HMF yield compared with transition metal chlorides including ZnCl2, CrCl3, CuCl2 and FeCl3. 1% (w/w) MgCl2 was the more favorable catalyst for 5-HMF production from agarose, and resulted in 40.7% 5-HMF yield but no levulinic acid or lactic acid at 200 oC, 35 min. The reaction pathways of agarose degradation catalyzed by MgCl2 were also discussed.

  1. Fenugreek hydrogel–agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection

    Energy Technology Data Exchange (ETDEWEB)

    Kestwal, Rakesh Mohan; Bagal-Kestwal, Dipali; Chiang, Been-Huang, E-mail: bhchiang@ntu.edu.tw

    2015-07-30

    A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel–agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10–20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel–agarose–acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples. - Highlights: • Acetylcholinesterase (AChE) dip-strip biosensor fabricated to detect carbamates. • AChE entrapped in fenugreek hydrogel–agarose matrix with gold nanoparticles (GNPs). • High enzyme retention efficiency (92%) and shelf life (half-life, 55 days). • Detection limits of carbofuran, oxamyl and methomyl: 2, 21 and 113 nM. • The biosensor had good testing capabilities to detect carbamates in food samples.

  2. Chondroitin sulfate-derivatized agarose beads: a new system for studying cation binding to glycosaminoglycans

    International Nuclear Information System (INIS)

    Hunter, G.K.

    1987-01-01

    Chondroitin sulfate (CS) has been covalently attached to aminoethyl-agarose beads in a carbodiimide-catalyzed reaction. In this process, an amide bond is formed between carboxylate groups on the glycosaminoglycan (GAG) and the primary amine groups of the beads. Under optimal conditions, up to 160 micrograms of CS is attached per milligram of beads. CS-agarose beads have been used to study Ca binding to GAGs. The beads are mixed with a solution containing CaCl 2 and 45 Ca and allowed to sediment under unit gravity. An aliquot of supernatant is then removed and 45 Ca activity is determined to quantitate remaining (free) Ca. Using this system, it was shown that CS binds approximately 0.7 Ca/disaccharide unit at saturation. Under the conditions used, the apparent association constant (KA) is approximately 14 mM. In principle, this derivatization protocol may be used to attach any proteoglycan or GAG (except keratan sulfate) to an insoluble support. CS-agarose beads provide a rapid, simple, and relatively artifact-free system for studying cation-GAG interactions

  3. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    International Nuclear Information System (INIS)

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s −1 ) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening

  4. Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

    Science.gov (United States)

    Maccari, Francesca; Volpi, Nicola

    2002-09-01

    We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

  5. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    Energy Technology Data Exchange (ETDEWEB)

    Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 11973-5000 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Teplitsky, Ella [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5215 (United States); Joshi, Karan [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); PEC University of Technology, Chandigarh (India); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Agarwal, Rakhi; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  6. Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing.

    Directory of Open Access Journals (Sweden)

    Xue Gong

    Full Text Available Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.

  7. Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction.

    Science.gov (United States)

    Kurien, B T; Kaufman, K M; Harley, J B; Scofield, R H

    2001-09-15

    A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit. Copyright 2001 Academic Press.

  8. Determination of glycated albumin using boronic acid-derived agarose beads on paper-based devices.

    Science.gov (United States)

    Ko, Euna; Tran, Van-Khue; Geng, Yanfang; Kim, Min Ki; Jin, Ga Hyun; Son, Seong Eun; Hur, Won; Seong, Gi Hun

    2018-01-01

    Self-monitoring of glycated albumin (GA), a useful glycemic marker, is an established method for preventing diabetes complications. Here, the paper-based lateral flow assay devices were developed for the sensitive detection of GA and the total human serum albumin (tHSA) in self-monitoring diabetes patients. Boronic acid-derived agarose beads were packed into a hole on a lateral flow channel. These well-coordinated agarose beads were used to capture GA through specific cis-diol interactions and to enhance the colorimetric signals by concentrating the target molecules. The devices exhibited large dynamic ranges (from 10  μ g/ml to 10 mg/ml for GA and from 10 mg/ml to 50 mg/ml for tHSA) and low detection limits (7.1  μ g/ml for GA and 4.7 mg/ml for tHSA), which cover the range of GA concentration in healthy plasma, which is 0.21-1.65 mg/ml (0.6%-3%). In determining the unknown GA concentrations in two commercial human plasma samples, the relative percentage difference between the values found by a standard ELISA kit and those found by our developed devices was 2.62% and 8.80%, which are within an acceptable range. The measurements of GA and tHSA were completed within 20 min for the total sample-to-answer diagnosis, fulfilling the demand for rapid analysis. Furthermore, the recovery values ranged from 99.4% to 110% in device accuracy tests. These results indicate that the developed paper-based device with boronic acid-derived agarose beads is a promising platform for GA and tHSA detection as applied to self-monitoring systems.

  9. Microneedle assisted micro-particle delivery from gene guns: experiments using skin-mimicking agarose gel.

    Science.gov (United States)

    Zhang, Dongwei; Das, Diganta B; Rielly, Chris D

    2014-02-01

    A set of laboratory experiments has been carried out to determine if micro-needles (MNs) can enhance penetration depths of high-speed micro-particles delivered by a type of gene gun. The micro-particles were fired into a model target material, agarose gel, which was prepared to mimic the viscoelastic properties of porcine skin. The agarose gel was chosen as a model target as it can be prepared as a homogeneous and transparent medium with controllable and reproducible properties allowing accurate determination of penetration depths. Insertions of various MNs into gels have been analysed to show that the length of the holes increases with an increase in the agarose concentration. The penetration depths of micro-particle were analysed in relation to a number of variables, namely the operating pressure, the particle size, the size of a mesh used for particle separation and the MN dimensions. The results suggest that the penetration depths increase with an increase of the mesh pore size, because of the passage of large agglomerates. As these particles seem to damage the target surface, then smaller mesh sizes are recommended; here, a mesh with a pore size of 178 μm was used for the majority of the experiments. The operating pressure provides a positive effect on the penetration depth, that is it increases as pressure is increased. Further, as expected, an application of MNs maximises the micro-particle penetration depth. The maximum penetration depth is found to increase as the lengths of the MNs increase, for example it is found to be 1272 ± 42, 1009 ± 49 and 656 ± 85 μm at 4.5 bar pressure for spherical micro-particles of 18 ± 7 μm diameter when we used MNs of 1500, 1200 and 750 μm length, respectively. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  10. Micropatterning of a stretchable conductive polymer using inkjet printing and agarose stamping

    DEFF Research Database (Denmark)

    Hansen, Thomas Steen; Hassager, Ole; Larsen, Niels Bent

    2007-01-01

    A highly conducting stretchable polymer material has been patterned using additive inkjet printing and by subtractive agarose stamping of a deactivation agent (hypochlorite). The material consisted of elastomeric polyurethane combined in an interpenetrating network with a conductive polymer, poly(3....... Inkjet printing of the material was only possible if a short-chain polyurethane was used as elastomer to overcome strain hardening at the neck of the droplets produced for printing. Reproducible line widths down to 200 μm could be achieved by inkjet printing. Both methods were used to fabricate test...

  11. Characterization of agarose as immobilization matrix model for a microbial biosensor

    Directory of Open Access Journals (Sweden)

    Pernetti Mimma

    2003-01-01

    Full Text Available Microbial biosensors are promising tools for the detection of specific substances in different fields, such as environmental, biomedical, food or agricultural. They allow rapid measurements, no need for complex sample preparation or specialized personnel and easy handling. In order to enhance the managing, miniaturization and stability of the biosensor and to prevent cell leaching, bacteria immobilization is desirable. A systematic characterization procedure to choose a suitable immobilization method and matrix, was proposed in this study. Physical properties, storage stability mass transport phenomena and biocompatibility were evaluated, employing agarose as the model matrix. Preliminary essays with bioluminescent bacteria detecting Tributyltin were also carried out.

  12. The preparation of low electroendosmosis agarose and its physico-chemical property

    Science.gov (United States)

    Hu, Rugui; Liu, Xiaolei; Liu, Li; Zhang, Quanbin; Zhang, Hong; Niu, Xizhen

    2007-10-01

    Studies on Gelidium amansii agar fractionations were carried out in this paper. Gelidium amansii agar was fractionated on DEAE-Cellulose, and four fractions were obtained sequentially. The fractions were analyzed on physical and chemical properties, and IR and 13C-NMR spectroscopy applied for elucidating the chemical structure. Among the four fractions obtained, water fraction measured up to the standard of low EEO agarose. The sulfate content, ash content, electroendosmosis and gel strength (1%) of water fraction were 0.16%, 0.34%, 0.12 and 1 130g/cm2 respectively, similar to those of the Sigma products.

  13. Studies of transferin polymorphism in Swedish cattle using agarose gel electrophoresis

    International Nuclear Information System (INIS)

    Liberg, P.; Carlstroem, G.

    1976-01-01

    The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with 59 Fe. Six existing phenotypes based on the alleles Tf(supA), Tf(supD) and Tf(supE) could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencis between the Swedish Red and White and the Swedish Friesian. (author)

  14. Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases.

    Science.gov (United States)

    Vecoli, C; Prevost, F E; Ververis, J J; Medeiros, A A; O'Leary, G P

    1983-08-01

    Plasmid-mediated beta-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 beta-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pI 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of beta-lactamases.

  15. Tailor-made cell patterning using a near-infrared-responsive composite gel composed of agarose and carbon nanotubes

    International Nuclear Information System (INIS)

    Koga, Haruka; Nakazawa, Kohji; Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi

    2013-01-01

    Micropatterning is useful for regulating culture environments. We developed a highly efficient near-infrared-(NIR)-responsive gel and established a new technique that enables cell patterning by NIR irradiation. As a new culture substratum, we designed a tissue culture plate that was coated with a composite gel composed of agarose and carbon nanotubes (CNTs). A culture plate coated with agarose only showed no response to NIR irradiation. In contrast, NIR laser irradiation induced heat generation by CNTs; this permitted local solation of the CNT/agarose gel, and consequently, selective cell-adhesive regions were exposed on the tissue culture plate. The solation area was controlled by the NIR intensity, magnification of the object lens and CNT concentration in the gel. Furthermore, we formed circular patterns of HeLa cells and linear patterns of 3T3 cells on the same culture plate through selective and stepwise NIR irradiation of the CNT/agarose gel, and we also demonstrated that individual 3T3 cells migrated along a linear path formed on the CNT/agarose gel by NIR irradiation. These results indicate that our technique is useful for tailor-made cell patterning of stepwise and/or complex cell patterns, which has various biological applications such as stepwise co-culture and the study of cell migration. (paper)

  16. Pravastatin Improves Glucose Regulation and Biocompatibility of Agarose Encapsulated Porcine Islets following Transplantation into Pancreatectomized Dogs

    Directory of Open Access Journals (Sweden)

    Lawrence S. Gazda

    2014-01-01

    Full Text Available The encapsulation of porcine islets is an attractive methodology for the treatment of Type I diabetes. In the current study, the use of pravastatin as a mild anti-inflammatory agent was investigated in pancreatectomized diabetic canines transplanted with porcine islets encapsulated in agarose-agarose macrobeads and given 80 mg/day of pravastatin (n=3 while control animals did not receive pravastatin (n=3. Control animals reached preimplant insulin requirements on days 18, 19, and 32. Pravastatin-treated animals reached preimplant insulin requirements on days 22, 27, and 50. Two animals from each group received a second macrobead implant: control animals remained insulin-free for 15 and 21 days (AUC = 3003 and 5078 mg/dL/24 hr days 1 to 15 and reached preimplant insulin requirements on days 62 and 131. Pravastatin treated animals remained insulin-free for 21 and 34 days (AUC = 1559 and 1903 mg/dL/24 hr days 1 to 15 and reached preimplant insulin requirements on days 38 and 192. Total incidence (83.3% versus 64.3% and total severity (22.7 versus 18.3 of inflammation on tissue surfaces were higher in the control group at necropsy. These findings support pravastatin therapy in conjunction with the transplantation of encapsulated xenogeneic islets for the treatment of diabetes mellitus.

  17. Use of a commercial agarose gel for analysis of urinary glycosaminoglycans in mucopolysaccharidoses

    Directory of Open Access Journals (Sweden)

    Ana Carolina Breier

    Full Text Available ABSTRACT Mucopolysaccharidoses (MPS are a group of inherited metabolic disorders caused by deficiency of enzymes that degrade glycosaminoglycans (GAGs. Urinary excretion of GAGs is a common feature of MPS, and is considered their major biomarker. We aimed to adapt the GAG electrophoresis method to a commercial agarose gel which would be able to separate urinary GAGs in a simpler way with good sensitivity and reproducibility. Urine samples from patients previously diagnosed with MPS I, IV, and VI were used as electrophoretic standards. Samples from patients on enzyme replacement therapy (ERT were also assessed. Commercial agarose gel electrophoresis was effective, showing proper definition and separation of GAG bands. Detection sensitivity exceeded 0.1 µg and band reproducibility were consistent. GAG bands quantified in urine samples from patients on ERT correlated very strongly (correlation coefficient = 0.98 with total GAG concentrations. This application of gel electrophoresis demonstrates the possibility of monitoring patients with MPS treated with ERT by analyzing separately the GAGs excreted in urine. We suggest this process should be applied to MPS screening as well as to follow-up of patients on treatment.

  18. Agarose-gel electrophoresis for the quality assurance and purity of heparin formulations.

    Science.gov (United States)

    Volpi, Nicola; Buzzega, Dania

    2012-01-01

    The adulteration of raw heparin (Hep) with a synthetic oversulfated chondroitin sulfate (OSCS) not found in nature produced in 2007-2008 a global crisis giving rise to the development of additional, new and specific methods for its quality assurance and purity. In this study, a simple and sensitive agarose-gel electrophoresis method has been developed for the visualization of OSCS in Hep samples along with other natural glycosaminoglycans possibly present as "process-related impurities", in particular dermatan sulfate (DS) and chondroitin sulfate (CS). Agarose-gel electrophoresis under non-conventional conditions is able to separate OSCS from Hep with its two components, the slow-moving and fast-moving species, DS and CS by performing separation for 15 h (overnight) and under high voltage (100 mA, ∼200 V). Densitometric scanning enabled us to calculate a limit of detection of ∼0.5 μg OSCS with a linear behaviour from 0.1 to 5 μg, comparable to CS/DS. Contaminated samples from Hep manufacturers were analyzed and quantitative data were found comparable to previous studies. Due to its capacity to process many samples in a single run and to the equipment commonly available in laboratories, this analytical method would be suitable for the identification and quantification of contamination by other polysaccharides, in particular OSCS and DS, within Hep preparations and formulations. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Fabrication and Optimization of a PAGATA Gel Dosimeter: Increasing the Melting Point of the PAGAT Gel Dosimeter with Agarose Additive

    Directory of Open Access Journals (Sweden)

    Bakhtiar Azadbakht

    2010-12-01

    Full Text Available Introduction: The PAGAT polymer gel dosimeter melts at 30 ˚C and even at room temperature during the summer, so it needs to be kept in a cool place such as a refrigerator. To increase the stability of the PAGAT gel, different amounts of agarose were added to the PAGAT gel composition and the PAGATA gel was manufactured. Material and Methods: The PAGATA gel vials were irradiated using a Co-60 machine. Then, the samples were evaluated using a 1.5 T Siemens MRI scanner. The ingredients of the PAGATA normoxic gel dosimeter were 4.5% N-N' methylen-bis-acrylamide, 4.5% acrylamide, 4.5% gelatine, 5 mM tetrakis (THPC, 0.01 mM hydroquinone (HQ, 0.5% agarose and 86% de-ionized water (HPLC. Results: Melting point and sensitivity of the PAGAT gel dosimeter with addition of 0.0, 0.3, 0.5, 1.0, 1.5 and 2.0% of agarose were measured, in which the melting points were increased to 30, 82, 86, 88, 89 and 90°C and their sensitivities found to be 0.113, 0.1059, 0.125, 0.122, 0.115 and 0.2  respectively. Discussion and Conclusions: Adding agarose increased the sensitivity and background R2 of the evaluated samples. The optimum amount of agarose was found to be 0.5% regarding these parameters and also the melting point of the gel dosimeter. A value of 0.5% agarose was found to be an optimum value considering the increase of sensitivity to 0.125 and melting point to 86°C but at the expense of increasing the background R2 to 4.530.

  20. Three-dimensional determination of absorbed dose by spectrophotometric analysis of ferrous-sulphate agarose gel

    International Nuclear Information System (INIS)

    Gambarini, G.; Gomarasca, G.; Marchesini, R.; Pecci, A.; Pirola, L.; Tomatis, S.

    1999-01-01

    We describe a technique to obtain three-dimensional (3-D) imaging of an absorbed dose by optical transmittance measurements of phantoms composed by agarose gel in which a ferrous sulphate and xylenol orange solution are incorporated. The analysis of gel samples is performed by acquiring transmittance images with a system based on a CCD camera provided with an interference filter matching the optical absorption peak of interest. The proposed technique for 3-D measurements of an absorbed dose is based on the imaging of phantoms composed of sets of properly piled up gel slices. The slice thickness was optimized in order to obtain a good image contrast as well as a good in-depth spatial resolution. To test the technique, a phantom has been irradiated with a collimated γ-beam and then analysed. Proper software was adapted in order to visualise the images of all slices and to attain the 2-D profiles of the dose absorbed by each slice

  1. UTILITY OF FUNCTIONALIZED AGAROSE NANOPARTICLES IN HYDROLYZING LACTOSE IN BATCH REACTORS FOR DAIRY INDUSTRIES

    Directory of Open Access Journals (Sweden)

    Shakeel Ahmed Ansari

    Full Text Available The present study investigates the synthesis of agarose nanoparticles (ANPs and its surface modification by galactose for the immobilization of β-galactosidase. Galactose modified ANPs retained 91% enzyme activity upon immobilization. Optimum pH (4.5 and temperature (50 ºC remains unchanged after immobilization. However, immobilized enzyme retained greater catalytic activity against lower and higher, pH and temperature ranges. Immobilized β-galactosidase retained 89% biocatalytic activity even at 4% galactose concentration as compared to enzyme in solution, and exhibited 81% activity even after seventh repeated uses. Immobilized enzyme hydrolyzed greater amount of lactose at higher temperatures as compared to β-galactosidase in solution, thereby suggesting its potential application in obtaining lactose-free dairy products at large scale.

  2. Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads.

    Science.gov (United States)

    Bental, M; Pick, U; Avron, M; Degani, H

    1990-02-22

    A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.

  3. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    International Nuclear Information System (INIS)

    Vetcher, Alexandre A; Srinivasan, Srimeenakshi; Vetcher, Ivan A; Abramov, Semen M; Kozlov, Mikhail; Baughman, Ray H; Levene, Stephen D

    2006-01-01

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique

  4. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Vetcher, Alexandre A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Srinivasan, Srimeenakshi [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Vetcher, Ivan A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Abramov, Semen M [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Kozlov, Mikhail [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Baughman, Ray H [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Levene, Stephen D [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States)

    2006-08-28

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

  5. Comparative assessment of intrinsic mechanical stimuli on knee cartilage and compressed agarose constructs.

    Science.gov (United States)

    Completo, A; Bandeiras, C; Fonseca, F

    2017-06-01

    A well-established cue for improving the properties of tissue-engineered cartilage is mechanical stimulation. However, the explicit ranges of mechanical stimuli that correspond to favorable metabolic outcomes are elusive. Usually, these outcomes have only been associated with the applied strain and frequency, an oversimplification that can hide the fundamental relationship between the intrinsic mechanical stimuli and the metabolic outcomes. This highlights two important key issues: the firstly is related to the evaluation of the intrinsic mechanical stimuli of native cartilage; the second, assuming that the intrinsic mechanical stimuli will be important, deals with the ability to replicate them on the tissue-engineered constructs. This study quantifies and compares the volume of cartilage and agarose subjected to a given magnitude range of each intrinsic mechanical stimulus, through a numerical simulation of a patient-specific knee model coupled with experimental data of contact during the stance phase of gait, and agarose constructs under direct-dynamic compression. The results suggest that direct compression loading needs to be parameterized with time-dependence during the initial culture period in order to better reproduce each one of the intrinsic mechanical stimuli developed in the patient-specific cartilage. A loading regime which combines time periods of low compressive strain (5%) and frequency (0.5Hz), in order to approach the maximal principal strain and fluid velocity stimulus of the patient-specific cartilage, with time periods of high compressive strain (20%) and frequency (3Hz), in order to approach the pore pressure values, may be advantageous relatively to a single loading regime throughout the full culture period. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

  6. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  7. No Evidence of Viral Transmission following Long-Term Implantation of Agarose Encapsulated Porcine Islets in Diabetic Dogs

    Directory of Open Access Journals (Sweden)

    Lawrence S. Gazda

    2014-01-01

    Full Text Available We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n=3 was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652 when compared to a first macrobead implantation (days 9, 21, and 21 in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

  8. Photothermal Microneedle Etching: Improved Three-Dimensional Microfabrication Method for Agarose Gel for Topographical Control of Cultured Cell Communities

    Science.gov (United States)

    Moriguchi, Hiroyuki; Yasuda, Kenji

    2006-08-01

    We have developed a new three-dimensional (3D) microfabrication method for agarose gel, photothermal microneedle etching (PTMNE), by means of an improved photothermal spot heating using a focused 1064 nm laser beam for melting a portion of the agarose layer at the tip of the microneedle, where a photoabsorbent chromium layer is coated to be heated. The advantage of this method is that it allows the 3D control of the melting topography within the thick agarose layer with a 2 μm resolution, whereas conventional photothermal etching can enable only two-dimensional (2D) control on the surface of the chip. By this method, we can form the spheroid clusters of particular cells from isolated single cells without any physical contact with other cells in other chambers, which is important for measuring the community effect of the cell group from isolated single cells. When we set single cancer cells in microchambers of 100 μm in diameter, formed in a 50-μm-thick agarose layer, we observed that they grew, divided, and formed spheroid clusters of cells in each microchamber. The result indicates the potential of this method to be a fundamental technique in the research of multicellular spherical clusters of cells for checking the community effect of cells in 3D structures, such as the permeabilities of chemicals and substrates into the cluster, which is complementary to conventional 2D dish cultivation and can contribute to the cell-based screening of drugs.

  9. Tuning mechanical performance of poly(ethylene glycol) and agarose interpenetrating network hydrogels for cartilage tissue engineering.

    Science.gov (United States)

    Rennerfeldt, Deena A; Renth, Amanda N; Talata, Zsolt; Gehrke, Stevin H; Detamore, Michael S

    2013-11-01

    Hydrogels are attractive for tissue engineering applications due to their incredible versatility, but they can be limited in cartilage tissue engineering applications due to inadequate mechanical performance. In an effort to address this limitation, our team previously reported the drastic improvement in the mechanical performance of interpenetrating networks (IPNs) of poly(ethylene glycol) diacrylate (PEG-DA) and agarose relative to pure PEG-DA and agarose networks. The goal of the current study was specifically to determine the relative importance of PEG-DA concentration, agarose concentration, and PEG-DA molecular weight in controlling mechanical performance, swelling characteristics, and network parameters. IPNs consistently had compressive and shear moduli greater than the additive sum of either single network when compared to pure PEG-DA gels with a similar PEG-DA content. IPNs withstood a maximum stress of up to 4.0 MPa in unconfined compression, with increased PEG-DA molecular weight being the greatest contributing factor to improved failure properties. However, aside from failure properties, PEG-DA concentration was the most influential factor for the large majority of properties. Increasing the agarose and PEG-DA concentrations as well as the PEG-DA molecular weight of agarose/PEG-DA IPNs and pure PEG-DA gels improved moduli and maximum stresses by as much as an order of magnitude or greater compared to pure PEG-DA gels in our previous studies. Although the viability of encapsulated chondrocytes was not significantly affected by IPN formulation, glycosaminoglycan (GAG) content was significantly influenced, with a 12-fold increase over a three-week period in gels with a lower PEG-DA concentration. These results suggest that mechanical performance of IPNs may be tuned with partial but not complete independence from biological performance of encapsulated cells. © 2013 Elsevier Ltd. All rights reserved.

  10. Analysis of surface properties of fixed and live cells using derivatized agarose beads.

    Science.gov (United States)

    Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

    2002-01-01

    A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

  11. Mechanical characterisation of agarose-based chromatography resins for biopharmaceutical manufacture.

    Science.gov (United States)

    Nweke, Mauryn C; McCartney, R Graham; Bracewell, Daniel G

    2017-12-29

    Mechanical characterisation of agarose-based resins is an important factor in ensuring robust chromatographic performance in the manufacture of biopharmaceuticals. Pressure-flow profiles are most commonly used to characterise these properties. There are a number of drawbacks with this method, including the potential need for several re-packs to achieve the desired packing quality, the impact of wall effects on experimental set up and the quantities of chromatography media and buffers required. To address these issues, we have developed a dynamic mechanical analysis (DMA) technique that characterises the mechanical properties of resins based on the viscoelasticity of a 1ml sample of slurry. This technique was conducted on seven resins with varying degrees of mechanical robustness and the results were compared to pressure-flow test results on the same resins. Results show a strong correlation between the two techniques. The most mechanically robust resin (Capto Q) had a critical velocity 3.3 times higher than the weakest (Sepharose CL-4B), whilst the DMA technique showed Capto Q to have a slurry deformation rate 8.3 times lower than Sepharose CL-4B. To ascertain whether polymer structure is indicative of mechanical strength, scanning electron microscopy images were also used to study the structural properties of each resin. Results indicate that DMA can be used as a small volume, complementary technique for the mechanical characterisation of chromatography media. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

  12. Stabilization of Candida antarctica Lipase B (CALB Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI

    Directory of Open Access Journals (Sweden)

    Sara Peirce

    2016-06-01

    Full Text Available Lipase B from Candida antarctica (CALB was immobilized on octyl agarose (OC and physically modified with polyethyleneimine (PEI in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

  13. Model creation of moving redox reaction boundary in agarose gel electrophoresis by traditional potassium permanganate method.

    Science.gov (United States)

    Xie, Hai-Yang; Liu, Qian; Li, Jia-Hao; Fan, Liu-Yin; Cao, Cheng-Xi

    2013-02-21

    A novel moving redox reaction boundary (MRRB) model was developed for studying electrophoretic behaviors of analytes involving redox reaction on the principle of moving reaction boundary (MRB). Traditional potassium permanganate method was used to create the boundary model in agarose gel electrophoresis because of the rapid reaction rate associated with MnO(4)(-) ions and Fe(2+) ions. MRB velocity equation was proposed to describe the general functional relationship between velocity of moving redox reaction boundary (V(MRRB)) and concentration of reactant, and can be extrapolated to similar MRB techniques. Parameters affecting the redox reaction boundary were investigated in detail. Under the selected conditions, good linear relationship between boundary movement distance and time were obtained. The potential application of MRRB in electromigration redox reaction titration was performed in two different concentration levels. The precision of the V(MRRB) was studied and the relative standard deviations were below 8.1%, illustrating the good repeatability achieved in this experiment. The proposed MRRB model enriches the MRB theory and also provides a feasible realization of manual control of redox reaction process in electrophoretic analysis.

  14. Use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels

    Energy Technology Data Exchange (ETDEWEB)

    McMichael, J C; Greisiger, L M; Millman, I [Institute for Cancer Research, Philadelphia, PA (USA). Fox Chase Cancer Center

    1981-08-28

    Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). /sup 125/I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 ..mu..l or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, the authors observed two bands of HBsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck hepatitis virus surface antigen (WHsAg).

  15. Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

    Science.gov (United States)

    Jimenez, Maria S; Luque-Alled, Jose M; Gomez, Teresa; Castillo, Juan R

    2016-05-01

    Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

    Science.gov (United States)

    Serwer, Philip; Wright, Elena T.

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

  17. Application of native agarose gel electrophoresis of serum proteins in veterinary diagnostics

    Directory of Open Access Journals (Sweden)

    Jania Bartosz

    2016-12-01

    Full Text Available Electrophoretic techniques, used to separate mixtures of electrically charged particles, are widely used in science. One of these techniques, native protein electrophoresis in an agarose gel, is applied in human and veterinary medicine. Changes in the proportions of individual protein fractions correspond to significant changes in the physiology of the body. Although the pattern obtained by electrophoretic separation rarely indicates a specific disease, it provides valuable information for the differential diagnosis. Decades of research on the types of patterns obtained in the case of particular diseases have led to the accumulation of substantial knowledge. The paper presents the available information on this topic. Serum protein electrophoresis is recommended in cases of increased levels of total protein in order to reveal the nature of the process. The basic information which can be obtained from electrophoretic separation includes the immune status of the organism. Both increased antigenic stimulation and immunodeficiency are clearly visible in electropherograms. Moreover, the level of heterogeneity of the corresponding protein fractions can help to distinguish between infectious diseases and cancer - multiple myeloma - the latter producing a homogeneous immunoglobulin fraction. Analysis of other protein fractions helps to detect or confirm an ongoing inflammatory process and provides information regarding liver function. Even when the concentration of total protein is within the reference range, this analysis can be recommended as a basic laboratory test.

  18. Agarose gel electrophoresis and polyacrylamide gel electrophoresis for visualization of simple sequence repeats.

    Science.gov (United States)

    Anderson, James; Wright, Drew; Meksem, Khalid

    2013-01-01

    In the modern age of genetic research there is a constant search for ways to improve the efficiency of plant selection. The most recent technology that can result in a highly efficient means of selection and still be done at a low cost is through plant selection directed by simple sequence repeats (SSRs or microsatellites). The molecular markers are used to select for certain desirable plant traits without relying on ambiguous phenotypic data. The best way to detect these is the use of gel electrophoresis. Gel electrophoresis is a common technique in laboratory settings which is used to separate deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) by size. Loading DNA and RNA onto gels allows for visualization of the size of fragments through the separation of DNA and RNA fragments. This is achieved through the use of the charge in the particles. As the fragments separate, they form into distinct bands at set sizes. We describe the ability to visualize SSRs on slab gels of agarose and polyacrylamide gel electrophoresis.

  19. Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins.

    Science.gov (United States)

    McCudden, Christopher R; Mathews, Stephanie P; Hainsworth, Shirley A; Chapman, John F; Hammett-Stabler, Catherine A; Willis, Monte S; Grenache, David G

    2008-03-01

    The objective of this study was to compare gel- and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

  20. DNA agarose gel electrophoresis for antioxidant analysis: Development of a quantitative approach for phenolic extracts.

    Science.gov (United States)

    Silva, Sara; Costa, Eduardo M; Vicente, Sandra; Veiga, Mariana; Calhau, Conceição; Morais, Rui M; Pintado, Manuela E

    2017-10-15

    Most of the fast in vitro assays proposed to determine the antioxidant capacity of a compound/extract lack either biological context or employ complex protocols. Therefore, the present work proposes the improvement of an agarose gel DNA electrophoresis in order to allow for a quantitative estimation of the antioxidant capacity of pure phenolic compounds as well as of a phenolic rich extract, while also considering their possible pro-oxidant effects. The result obtained demonstrated that the proposed method allowed for the evaluation of the protection of DNA oxidation [in the presence of hydrogen peroxide (H 2 O 2 ) and an H 2 O 2 /iron (III) chloride (FeCl 3 ) systems] as well as for the observation of pro-oxidant activities, with the measurements registering interclass correlation coefficients above 0.9. Moreover, this method allowed for the characterization of the antioxidant capacity of a blueberry extract while demonstrating that it had no perceived pro-oxidant effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.

    Science.gov (United States)

    Serwer, Philip; Wright, Elena T

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. In vivo remineralization of dentin using an agarose hydrogel biomimetic mineralization system

    Science.gov (United States)

    Han, Min; Li, Quan-Li; Cao, Ying; Fang, Hui; Xia, Rong; Zhang, Zhi-Hong

    2017-02-01

    A novel agarose hydrogel biomimetic mineralization system loaded with calcium and phosphate was used to remineralize dentin and induce the oriented densely parallel packed HA layer on defective dentin surface in vivo in a rabbit model. Firstly, the enamel of the labial surface of rabbits’ incisor was removed and the dentin was exposed to oral environment. Secondly, the hydrogel biomimetic mineralization system was applied to the exposed dentin surface by using a custom tray. Finally, the teeth were extracted and evaluated by scanning electron microscopy, X-ray diffraction, and nanoindentation test after a certain time of mineralization intervals. The regenerated tissue on the dentin surface was composed of highly organised HA crystals. Densely packed along the c axis, these newly precipitated HA crystals were perpendicular to the underlying dental surface with a tight bond. The demineralized dentin was remineralized and dentinal tubules were occluded by the grown HA crystals. The nanohardness and elastic modulus of the regenerated tissue were similar to natural dentin. The results indicated a potential clinical use for repairing dentin-exposed related diseases, such as erosion, wear, and dentin hypersensitivity.

  3. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

    Full Text Available OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05. The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05, indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

  4. Improved methods for the fluorographic detection of weak β-emitting radioisotopes in agarose and acrylamide gel electrophoresis media

    International Nuclear Information System (INIS)

    Pulleyblank, D.E.; Booth, G.M.

    1981-01-01

    The use of acetic acid as a solvent for diphenyloxazole (PPO) in fluorographic procedures has been investigated. It is demonstrated to be superior to both dimethyl sulfoxide and methanol with respect to its suitability in both agarose and acrylamide gel electrophoresis systems. In addition, a method has been developed for impregnating fragile gels such as those used for immunodiffusion with PPO in preparation for fluorography. (Auth.)

  5. Hollow agarose microneedle with silver coating for intradermal surface-enhanced Raman measurements: a skin-mimicking phantom study

    Science.gov (United States)

    Yuen, Clement; Liu, Quan

    2015-06-01

    Human intradermal components contain important clinical information beneficial to the field of immunology and disease diagnosis. Although microneedles have shown great potential to act as probes to break the human skin barrier for the minimally invasive measurement of intradermal components, metal microneedles that include stainless steel could cause the following problems: (1) sharp waste production, and (2) contamination due to reuse of microneedles especially in developing regions. In this study, we fabricate agarose microneedles coated with a layer of silver (Ag) and demonstrate their use as a probe for the realization of intradermal surface-enhanced Raman scattering measurements in a set of skin-mimicking phantoms. The Ag-coated agarose microneedle quantifies a range of glucose concentrations from 5 to 150 mM inside the skin phantoms with a root-mean-square error of 5.1 mM within 10 s. The needle is found enlarged by 53.9% after another 6 min inside the phantom. The shape-changing capability of this agarose microneedle ensures that the reuse of these microneedles is impossible, thus avoiding sharp waste production and preventing needle contamination, which shows the great potential for safe and effective needle-based measurements.

  6. Fabrication of agarose concave petridish for 3D-culture microarray method for spheroids formation of hepatic cells.

    Science.gov (United States)

    Zhang, Binbin; Li, Yang; Wang, Gaoshang; Jia, Zhidong; Li, Haiyan; Peng, Qing; Gao, Yi

    2018-04-19

    Liver is one of the most important organ in the body. But there are many limitations about liver transplantation for liver failure. It is quite important to develop the xenogeneic biological liver for providing an alternation to transplantation or liver regeneration. In this paper, we proposed a method to construct a novel kind of agarose 3D-culture concave microwell array for spheroids formation of hepatic cells. Using the 3D printing method, the microwell array was fabricated with an overall size of 6.4 mm × 6.4 mm, containing 121 microwells with 400 μm width/400 μm thickness. By exploiting the Polydimethylsiloxane (PDMS) membranes as a bridge, we finally fabricated the agarose one. We co-cultured three types of liver cells with bionics design in the microwell arrays. Using the methods described above, the resulting co-formed hepatocyte spheroids maintained the high viability and stable liver-specific functions. This engineered agarose concave microwell array could be a potentially useful tool for forming the elements for biological liver support. After developing the complete system, we also would consider to scale up the application of this system. It will be not only applied to the therapy of human organ damage, but also to the development of disease models and drug screening models.

  7. In Situ Observations of Thermoreversible Gelation and Phase Separation of Agarose and Methylcellulose Solutions under High Pressure.

    Science.gov (United States)

    Kometani, Noritsugu; Tanabe, Masahiro; Su, Lei; Yang, Kun; Nishinari, Katsuyoshi

    2015-06-04

    Thermoreversible sol-gel transitions of agarose and methylcellulose (MC) aqueous solutions on isobaric cooling or heating under high pressure up to 400 MPa have been investigated by in situ observations of optical transmittance and falling-ball experiments. For agarose, which undergoes the gelation on cooling, the application of pressure caused a gradual rise in the cloud-point temperature over the whole pressure range examined, which is almost consistent with the pressure dependence of gelling temperature estimated by falling-ball experiments, suggesting that agarose gel is stabilized by compression and that the gelation occurs nearly in parallel with phase separation under ambient and high-pressure conditions. For MC, which undergoes the gelation on heating, the cloud-point temperature showed a slight rise with an initial elevation of pressure up to ∼150 MPa, whereas it showed a marked depression above 200 MPa. In contrast, the gelling temperature of MC, which is nearly identical to the cloud-point temperature at ambient pressure, showed a monotonous rise with increasing pressure up to 350 MPa, which means that MC undergoes phase separation prior to gelation on heating under high pressure above 200 MPa. Similar results were obtained for the melting process of MC gel on cooling. The unique behavior of the sol-gel transition of MC under high pressure has been interpreted in terms of the destruction of hydrophobic hydration by compression.

  8. Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

    Science.gov (United States)

    Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

    2016-06-14

    Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

  9. IPN hydrogel nanocomposites based on agarose and ZnO with antifouling and bactericidal properties

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jingjing, E-mail: jjwang1@hotmail.com; Hu, Hongkai; Yang, Zhonglin; Wei, Jun; Li, Juan

    2016-04-01

    Nanocomposite hydrogels with interpenetrating polymer network (IPN) structure based on poly(ethylene glycol) methyl ether methacrylate modified ZnO (ZnO-PEGMA) and 4-azidobenzoic agarose (AG-N{sub 3}) were prepared by a one-pot strategy under UV irradiation. The hydrogels exhibited a highly macroporous spongelike structure, and the pore size decreased with the increase of the ZnO-PEGMA content. Due to the entanglement and favorable interactions between the two crosslinked networks, the IPN hydrogels exhibited excellent mechanical strength and light transmittance. The maximum compressive and tensile strengths of the IPN hydrogels reached 24.8 and 1.98 MPa respectively. The transparent IPN hydrogels transmitted more than 85% of visible light at all wavelengths (400–800 nm). The IPN hydrogels exhibited anti-adhesive property towards Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), and the bactericidal activity increased with the ZnO-PEGMA content. The incorporation of ZnO-PEGMA did not reduce the biocompatibility of the IPN hydrogels and all the IPN nanocomposites showed negligible cytotoxicity. The present study not only provided a facile method for preparing hydrogel nanocomposites with IPN structure but also developed a new hydrogel material which might be an excellent candidate for wound dressings. - Highlights: • IPN hydrogel nanocomposites were prepared by a one-pot strategy. • The maximum compressive and tensile strengths reached 24.8 and 1.98 MPa. • IPN hydrogels displayed excellent antibacterial activity and cytocompatibility. • This study provided a facile method for preparing IPN hydrogel nanocomposites.

  10. Effect of saccharide additives on response of ferrous-agarose-xylenol orange radiotherapy gel dosimeters

    International Nuclear Information System (INIS)

    Healy, B.J.; Zahmatkesh, M.H.; Nitschke, K.N.; Baldock, C.

    2003-01-01

    Glucose, sucrose, starch, and locust bean gum have been used as additives to the ferrous-agarose-xylenol orange (FAX) gel dosimeter. The saccharide enhanced dosimeters were found to have a higher dose sensitivity over a standard FAX gel as measured by both optical density change and magnetic resonance imaging (MRI). With optical density measurement, OD-dose sensitivity increases were up to 55% for glucose, 122% for sucrose and 43% for starch, while locust bean gum did not give a consistent response. With MRI, R 1 -dose sensitivity increases were up to 178% with sucrose addition. The FAX gel with sucrose was studied in greatest detail. The OD-dose sensitivity dependence on cooling rate was reduced for the sucrose FAX gel over the standard FAX gel, which has significant implications for uniform dose sensitivity in large gel phantoms. The thermal oxidation rate in the sucrose FAX gel was up to 2.3 times higher than in the standard gel. The OD-dose sensitivity of oxygenated sucrose FAX gels was 4.3 times greater than standard FAX gels, while continued enhancement in OD-dose sensitivity with increased sucrose concentrations beyond 2.0 g/l was found only for the oxygenated sucrose FAX gels. Both the molar absorption coefficient of the ferric ion-xylenol orange complex at 543 nm and gel pH were not affected by the presence of sucrose, with the implication that the higher OD-dose sensitivity of gels with saccharides is due to increased chain reaction production of ferric ions

  11. Dynamic compression of chondrocyte-agarose constructs reveals new candidate mechanosensitive genes.

    Directory of Open Access Journals (Sweden)

    Carole Bougault

    Full Text Available Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK pathways and Smad2/3 (members of the canonical transforming growth factor (TGF-β pathways. A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how

  12. Recording of radiation-induced optical density changes in doped agarose gels with a CCD camera

    International Nuclear Information System (INIS)

    Tarte, B.J.; Jardine, P.A.; Van Doorn, T.

    1996-01-01

    Full text: Spatially resolved dose measurement with iron-doped agarose gels is continuing to be investigated for applications in radiotherapy dosimetry. It has previously been proposed to use optical methods, rather than MRI, for dose measurement with such gels and this has been investigated using a spectrophotometer (Appleby A and Leghrouz A, Med Phys, 18:309-312, 1991). We have previously studied the use of a pencil beam laser for such optical density measurement of gels and are currently investigating charge-coupled devices (CCD) camera imaging for the same purpose but with the advantages of higher data acquisition rates and potentially greater spatial resolution. The gels used in these studies were poured, irradiated and optically analysed in Perspex casts providing gel sections 1 cm thick and up to 20 cm x 30 cm in dimension. The gels were also infused with a metal indicator dye (xylenol orange) to render the radiation induced oxidation of the iron in the gel sensitive to optical radiation, specifically in the green spectral region. Data acquisition with the CCD camera involved illumination of the irradiated gel section with a diffuse white light source, with the light from the plane of the gel section focussed to the CCD array with a manual zoom lens. The light was also filtered with a green colour glass filter to maximise the contrast between unirradiated and irradiated gels. The CCD camera (EG and G Reticon MC4013) featured a 1024 x 1024 pixel array and was interfaced to a PC via a frame grabber acquisition board with 8 bit resolution. The performance of the gel dosimeter was appraised in mapping of physical and dynamic wedged 6 MV X-ray fields. The results from the CCD camera detection system were compared with both ionisation chamber data and laser based optical density measurements of the gels. Cross beam profiles were extracted from each measurement system at a particular depth (eg. 2.3 cm for the physical wedge field) for direct comparison. A

  13. Preparation of a novel pH optical sensor using orange (II) based on agarose membrane as support.

    Science.gov (United States)

    Heydari, Rouhollah; Hosseini, Mohammad; Amraei, Ahmadreza; Mohammadzadeh, Ali

    2016-04-01

    A novel and cost effective optical pH sensor was prepared using covalent immobilization of orange (II) indicator on the agarose membrane as solid support. The fabricated optical sensor was fixed into a sample holder of a spectrophotometer instrument for pH monitoring. Variables affecting sensor performance including pH of dye bonding to agarose membrane and dye concentration were optimized. The sensor responds to the pH changes in the range of 3.0-10.0 with a response time of 2.0 min and appropriate reproducibility (RSD ≤ 0.9%). No significant variation was observed on sensor response after increasing the ionic strength in the range of 0.0-0.5M of sodium chloride. Determination of pH using the proposed optical sensor is quick, simple, inexpensive, selective and sensitive in the pH range of 3.0-10.0. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    Science.gov (United States)

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  15. Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

    Science.gov (United States)

    Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

    2017-12-21

    High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

  16. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.

    Science.gov (United States)

    Sanderson, Brian A; Araki, Naoko; Lilley, Jennifer L; Guerrero, Gilberto; Lewis, L Kevin

    2014-06-01

    Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Agarose hydrogel induced MCF-7 and BMG-1 cell line progressive 3D and 3D revert cultures.

    Science.gov (United States)

    Subramaniyan, Aishwarya; Ravi, Maddaly

    2018-04-01

    3D culture systems have enhanced the utility of cancer cell lines as they are considered closer to the in vivo systems. A variety of changes are induced in cells cultured in 3D systems; an apparent and striking feature being the spontaneous acquisition of distinct morphological entities. 3D reverts (3DRs) can be obtained by introducing 3D aggregates in scaffold/matrix-free culture units. It could be seen that the two cell lines used in this study exhibited differences in 3DR structures, though both were cultured on agarose hydrogels. Also, differences in 3DR formation, growth and survival were different. While 3D aggregates of several cell lines have been reported for a variety of studies, there are no studies that describe or utilize 3DRs. 3DRs can provide insights into complex events that can occur in cancer cells; especially as material to study metastasis, migration, and invasion. © 2017 Wiley Periodicals, Inc.

  18. Evaluation of the friction coefficient, the radial stress, and the damage work during needle insertions into agarose gels.

    Science.gov (United States)

    Urrea, Fabián A; Casanova, Fernando; Orozco, Gustavo A; García, José J

    2016-03-01

    Agarose hydrogels have been extensively used as a phantom material to mimic the mechanical behavior of soft biological tissues, e.g. in studies aimed to analyze needle insertions into the organs producing tissue damage. To better predict the radial stress and damage during needle insertions, this study was aimed to determine the friction coefficient between the material of commercial catheters and hydrogels. The friction coefficient, the tissue damage and the radial stress were evaluated at 0.2, 1.8, and 10mm/s velocities for 28, 30, and 32 gauge needles of outer diameters equal to 0.36, 0.31, and 0.23mm, respectively. Force measurements during needle insertions and retractions on agarose gel samples were used to analyze damage and radial stress. The static friction coefficient (0.295±0.056) was significantly higher than the dynamic (0.255±0.086). The static and dynamic friction coefficients were significantly smaller for the 0.2mm/s velocity compared to those for the other two velocities, and there was no significant difference between the friction coefficients for 1.8 and 10mm/s. Radial stress averages were 131.2±54.1, 248.3±64.2, and 804.9±164.3Pa for the insertion velocity of 0.2, 1.8, and 10mm/s, respectively. The radial stress presented a tendency to increase at higher insertion velocities and needle size, which is consistent with other studies. However, the damage work did not show to be a good predictor of tissue damage, which appears to be due to simplifications in the analytical model. Differently to other approaches, the method proposed here based on radial stress may be extended in future studies to quantity tissue damage in vivo along the entire needle track. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  20. Analysis of Endonuclease R·EcoRI Fragments of DNA from Lambdoid Bacteriophages and Other Viruses by Agarose-Gel Electrophoresis

    Science.gov (United States)

    Helling, Robert B.; Goodman, Howard M.; Boyer, Herbert W.

    1974-01-01

    By means of agarose-gel electrophoresis, endonuclease R·EcoRI-generated fragments of DNA from various viruses were separated, their molecular weights were determined, and complete or partial fragment maps for lambda, φ80, and hybrid phages were constructed. Images PMID:4372397

  1. Agarose cell block technique as a complementary method in the diagnosis of fungal osteomyelitis in a dog

    Directory of Open Access Journals (Sweden)

    N.S. Rocha

    2012-04-01

    Full Text Available A 7-year-old Labrador Retriever female dog presenting left forelimb lameness for one day was admitted to the Veterinary Hospital (UNESP-Botucatu for clinical evaluation. Several tests, including blood and image analysis, microbiological culture and cytology of lytic areas of affected bone were made in order to establish a diagnosis. Serum biochemical profile revealed increased levels of liver enzymes, plasma globulin, creatine kinase (CK and calcium. Hemogram revealed anemia and leukocytosis; left humerus image analysis revealed an osteolytic lesion and cytology revealed a suppurative periostitis. Differential diagnosis was a nonspecific infectious inflammatory process or osteosarcoma. Since it was not possible to achieve a definitive diagnosis and there was a highly suspicious for an infectious agent, an agarose cell block of the bone marrow fine-needle aspiration was made. The cytological examination of cell block presented similar findings as described previously. However, additional stains including periodic acid-Schiff (PAS were positive for fungal hyphae, which rendered a diagnosis of fungal osteomyelitis due to Aspergillus spp. This case report illustrates an uncommon cause of osteomyelitis for breed that was diagnosed by an underused method in veterinary medicine.

  2. BDNF gene delivery within and beyond templated agarose multi-channel guidance scaffolds enhances peripheral nerve regeneration

    Science.gov (United States)

    Gao, Mingyong; Lu, Paul; Lynam, Dan; Bednark, Bridget; Campana, W. Marie; Sakamoto, Jeff; Tuszynski, Mark

    2016-12-01

    Objective. We combined implantation of multi-channel templated agarose scaffolds with growth factor gene delivery to examine whether this combinatorial treatment can enhance peripheral axonal regeneration through long sciatic nerve gaps. Approach. 15 mm long scaffolds were templated into highly organized, strictly linear channels, mimicking the linear organization of natural nerves into fascicles of related function. Scaffolds were filled with syngeneic bone marrow stromal cells (MSCs) secreting the growth factor brain derived neurotrophic factor (BDNF), and lentiviral vectors expressing BDNF were injected into the sciatic nerve segment distal to the scaffold implantation site. Main results. Twelve weeks after injury, scaffolds supported highly linear regeneration of host axons across the 15 mm lesion gap. The incorporation of BDNF-secreting cells into scaffolds significantly increased axonal regeneration, and additional injection of viral vectors expressing BDNF into the distal segment of the transected nerve significantly enhanced axonal regeneration beyond the lesion. Significance. Combinatorial treatment with multichannel bioengineered scaffolds and distal growth factor delivery significantly improves peripheral nerve repair, rivaling the gold standard of autografts.

  3. [Molecular imaging of thrombus with microbubbles targeted to alphavbeta3-integrin using an agarose flow chamber model].

    Science.gov (United States)

    Hu, Guang-quan; Liu, Jian; Yang, Li; Yan, Yi; Wu, Jue-fei; Xie, Jia-jia; Cai, Jing-jing; Ji, Li-jing; Bin, Jian-ping

    2010-03-01

    To assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound. Targeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images. The VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (Pevaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.

  4. Spherical agarose-coated magnetic nanoparticles functionalized with a new salen for magnetic solid-phase extraction of uranyl ion

    International Nuclear Information System (INIS)

    Serenjeh, Fariba Nazari; Hashemi, Payman; Ghiasvand, Ali Reza; Naeimi, Hossein; Zakerzadeh, Elham

    2016-01-01

    The authors describe a method for magnetic solid phase extraction of uranyl ions from water samples. It is based on the use of spherical agarose-coated magnetic nanoparticles along with magnetic field agitation. The salen type Schiff base N,N’-bis(4-hydroxysalicylidene)-1,2-phenylenediamine was synthesized from resorcinol in two steps and characterized by infrared and nucleic magnetic resonance spectroscopies. The particles were then activated by an epichlorohydrin method and functionalized with the Schiff base which acts as a selective ligand for the extraction of UO 2 (II). Following preconcentration and elution with HCl, the ions were quantified by spectrophotometry using Arsenazo III as the indicator. The effects of pH value, ionic strength and amount of the adsorbent on the extraction of UO 2 (II) were optimized by a multivariate central composite design method. Six replicate analyses under optimized conditions resulted in a recovery of 96.6 % with a relative standard deviation of 3.4 % for UO 2 (II). The detection limit of the method (at a signal-to-noise ratio of 3σ) is 10 μg L -1 . The method was successfully applied to the determination of UO 2 (II) in spiked water samples. (author)

  5. First-in-Human Phase 1 Trial of Agarose Beads Containing Murine RENCA Cells in Advanced Solid Tumors

    Directory of Open Access Journals (Sweden)

    Barry H. Smith

    2016-01-01

    Full Text Available Purpose Agarose macrobeads containing mouse renal adenocarcinoma cells (RMBs release factors, suppressing the growth of cancer cells and prolonging survival in spontaneous or induced tumor animals, mediated, in part, by increased levels of myocyte-enhancing factor (MEF2D via EGFR-and AKT-signaling pathways. The primary objective of this study was to determine the safety of RMBs in advanced, treatment-resistant metastatic cancers, and then its efficacy (survival, which is the secondary objective. Methods Thirty-one patients underwent up to four intraperitoneal implantations of RMBs (8 or 16 macrobeads/kg via laparoscopy in this single-arm trial (FDA BB-IND 10091; NCT 00283075. Serial physical examinations, laboratory testing, and PET-CT imaging were performed before and three months after each implant. Results RMBs were well tolerated at both dose levels (mean 660.9 per implant. AEs were (Grade 1/2 with no treatment-related SAEs. Conclusion The data support the safety of RMB therapy in advanced-malignancy patients, and the preliminary evidence for their potential efficacy is encouraging. A Phase 2 efficacy trial is ongoing.

  6. Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique.

    Science.gov (United States)

    Gama, Fernanda Gomes Velasque; Santana, Aureo Evangelista; Filho, Eugênio de Campos; Nogueira, Cláudia Aparecida da Silva

    2007-03-01

    Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.

  7. High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

    Science.gov (United States)

    Cigan, Alexander D; Roach, Brendan L; Nims, Robert J; Tan, Andrea R; Albro, Michael B; Stoker, Aaron M; Cook, James L; Vunjak-Novakovic, Gordana; Hung, Clark T; Ateshian, Gerard A

    2016-06-14

    Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Electro-driven extraction of polar compounds using agarose gel as a new membrane: Determination of amino acids in fruit juice and human plasma samples.

    Science.gov (United States)

    Sedehi, Samira; Tabani, Hadi; Nojavan, Saeed

    2018-03-01

    In this work, polypropylene hollow fiber was replaced by agarose gel in conventional electro membrane extraction (EME) to develop a novel approach. The proposed EME method was then employed to extract two amino acids (tyrosine and phenylalanine) as model polar analytes, followed by HPLC-UV. The method showed acceptable results under optimized conditions. This green methodology outperformed conventional EME, and required neither organic solvents nor carriers. The effective parameters such as the pH values of the acceptor and the donor solutions, the thickness and pH of the gel, the extraction voltage, the stirring rate, and the extraction time were optimized. Under the optimized conditions (acceptor solution pH: 1.5; donor solution pH: 2.5; agarose gel thickness: 7mm; agarose gel pH: 1.5; stirring rate of the sample solution: 1000rpm; extraction potential: 40V; and extraction time: 15min), the limits of detection and quantification were 7.5ngmL -1 and 25ngmL -1 , respectively. The extraction recoveries were between 56.6% and 85.0%, and the calibration curves were linear with correlation coefficients above 0.996 over a concentration range of 25.0-1000.0ngmL -1 for both amino acids. The intra- and inter-day precisions were in the range of 5.5-12.5%, and relative errors were smaller than 12.0%. Finally, the optimized method was successfully applied to preconcentrate, clean up, and quantify amino acids in watermelon and grapefruit juices as well as a plasma sample, and acceptable relative recoveries in the range of 53.9-84.0% were obtained. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    Science.gov (United States)

    Zhong, Ruibo; Yuan, Ming; Gao, Haiyang; Bai, Zhijun; Guo, Jun; Zhao, Xinmin; Zhang, Feng

    2016-03-01

    Discrete biomolecule-nanoparticle (NP) conjugates play paramount roles in nanofabrication, in which the key is to get the precise molar extinction coefficient of NPs. By making best use of the gift from a specific separation phenomenon of agarose gel electrophoresis (GE), amphiphilic polymer coated NP with exact number of bovine serum albumin (BSA) proteins can be extracted and further experimentally employed to precisely calculate the molar extinction coefficient of the NPs. This method could further benefit the evaluation and extraction of any other dual-component NP-containing bio-conjugates.

  10. Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

    Directory of Open Access Journals (Sweden)

    Shailesh S. Sawant

    2017-02-01

    Full Text Available Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs in pure cultures of marine bacterium, Saccharophagus degradans 2-40 (Sde 2-40, its contaminant, Bacillus cereus, and a co-culture of these bacteria (Sde 2-40 and B. cereus degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight. The present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.

  11. Co-immobilization of cyclohexanone monooxygenase and glucose-6-phosphate dehydrogenase onto polyethylenimine-porous agarose polymeric composite using γ irradiation to use in biotechnological processes

    International Nuclear Information System (INIS)

    Atia, K.S.

    2005-01-01

    The co-immobilization of cyclohexanone monooxygenase (CHMO) and glucose-6-phosphate dehydrogenase (G6PDH) was optimized by completely coating, via covalent immobilization, the surface aldehyde groups of porous agarose (glyoxyl-agarose) with amine groups of polyethylenimine (PEI). The highest immobilization efficiency (∼87%) (activity of enzyme per amount of immobilized enzyme) was obtained with a CHMO/G6PDH ratio 2:1. The effects of different ratios of the support to the amount of enzymes (CHMO:G6PDH=2:1), the optimum incubation pH and the incubation time on the enzymatic activity of the enzymes were determined and found to be 5:1, 8.5 and 30 min, respectively. Subjecting the co-immobilized enzymes to doses of γ-radiation (5-100 kGy) resulted in complete loss in the activity of the free enzymes at a dose of 40 kGy, while the co-immobilized ones showed relatively high resistance to γ-radiation up to a dose of 50 kGy

  12. Measurement of the ferric diffusion coefficient in agarose and gelatine gels by utilization of the evolution of a radiation induced edge as reflected in relaxation rate images

    International Nuclear Information System (INIS)

    Pedersen, Torje V.; Olsen, Dag R.; Skretting, Arne

    1997-01-01

    A method has been developed to determine the diffusion coefficients of ferric ions in ferrous sulphate doped gels. A radiation induced edge was created in the gel, and two spin-echo sequences were used to acquire a pair of images of the gel at different points of time. For each of these image pairs, a longitudinal relaxation rate image was derived. From profiles through these images, the standard deviations of the Gaussian functions that characterize diffusion were determined. These data provided the basis for the determination of the ferric diffusion coefficients by two different methods. Simulations indicate that the use of single spin-echo images in this procedure may in some cases lead to a significant underestimation of the diffusion coefficient. The technique was applied to different agarose and gelatine gels that were prepared, irradiated and imaged simultaneously. The results indicate that the diffusion coefficient is lower in a gelatine gel than in an agarose gel. Addition of xylenol orange to a gelatine gel lowers the diffusion coefficient from 1.45 to 0.81 mm 2 h -1 , at the cost of significantly lower R 1 sensitivity. The addition of benzoic acid to the latter gel did not increase the R 1 sensitivity. (author) OK

  13. Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.

    2006-01-01

    Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an ag......Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation...... to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20...... degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments...

  14. Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels

    International Nuclear Information System (INIS)

    Pagratis, N.; Revel, H.R.

    1990-01-01

    Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription

  15. Evaluation of a capillary zone electrophoresis system versus a conventional agarose gel system for routine serum protein separation and monoclonal component typing.

    Science.gov (United States)

    Roudiere, L; Boularan, A M; Bonardet, A; Vallat, C; Cristol, J P; Dupuy, A M

    2006-01-01

    Capillary zone electrophoresis of serum proteins is increasingly gaining impact in clinical laboratories. During 2003, we compared the fully automated capillary electrophoresis (CE) system from Beckman (Paragon CZE 2000) with the method agarose gel electrophoresis Sebia (Hydrasis-Hyris, AGE). This new study focused on the evaluation of analytical performance and a comparison including 115 fresh routine samples (group A) and a series of 97 frozen pathologic sera with suspicion of monoclonal protein (group B). Coefficients of variation (CVs %) for the five classical protein fractions have been reported to be consistenly serum samples (group B), there were 90 in which we detected a monoclonal protein by immunofixation (IF) (immunosubtraction (IS) was not used). AGE and Paragon 2000 failed to detect 7 and 12 monoclonal proteins, respectively, leading to a concordance to 92% for AGE and 87% for Paragon 2000 for identifying electrophoretic abnormalities in this group. Beta-globulin abnormalities and M paraprotein were well detected with Paragon 2000. Only 81% (21 vs 26) of the gammopathies were immunotyped with IS by two readers blinded to the IF immunotype. The Paragon 2000 is a reliable alternative to conventional agarose gel electrophoresis combining the advantages of full automation (rapidity, ease of use and cost) with high analytical performance. Qualified interpretation of results requires an adaptation period which could further improve concordance between the methods. Recently, this CE system has been improved by the manufacturer (Beckman) concerning the migration buffer and detection of beta-globulin abnormalities.

  16. The effects of cyclic hydrostatic pressure on chondrogenesis and viability of human adipose- and bone marrow-derived mesenchymal stem cells in three-dimensional agarose constructs.

    Science.gov (United States)

    Puetzer, Jennifer; Williams, John; Gillies, Allison; Bernacki, Susan; Loboa, Elizabeth G

    2013-01-01

    This study investigates the effects of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3-D) agarose constructs maintained in a complete growth medium without soluble chondrogenic inducing factors. hASCs were seeded in 2% agarose hydrogels and exposed to 7.5 MPa CHP for 4 h per day at a frequency of 1 Hz for up to 21 days. On days 0, 7, 14, and 21, the expression levels of collagen II, Sox9, aggrecan, and cartilage oligomeric matrix protein (COMP) were examined by real-time reverse transcriptase-polymerase chain reaction analysis. Gene expression analysis found collagen II mRNA expression in only the CHP-loaded construct at day 14 and at no other time during the study. CHP-loaded hASCs exhibited upregulated mRNA expression of Sox9, aggrecan, and COMP at day 7 relative to unloaded controls, suggesting that CHP initiated chondrogenic differentiation of hASCs in a manner similar to human bone marrow-derived mesenchymal stem cells (hMSC). By day 14, however, loaded hASC constructs exhibited significantly lower mRNA expression of the chondrogenic markers than unloaded controls. Additionally, by day 21, the samples exhibited little measurable mRNA expression at all, suggesting a decreased viability. Histological analysis validated the lack of mRNA expression at day 21 for both the loaded and unloaded control samples with a visible decrease in the cell number and change in morphology. A comparative study with hASCs and hMSCs further examined long-term cell viability in 3-D agarose constructs of both cell types. Decreased cell metabolic activity was observed throughout the 21-day experimental period in both the CHP-loaded and control constructs of both hMSCs and hASCs, suggesting a decrease in cell metabolic activity, alluding to a decrease in cell viability. This suggests that a 2% agarose hydrogel may not optimally support hASC or hMSC viability in a complete growth medium in the

  17. Calibration of denaturing agarose gels for molecular weight estimation of DNA: size determination of the single-stranded genomes of parvoviruses

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, C.E. (Oak Ridge National Lab., TN); Schmoyer, R.L.; Bates, R.C.; Mitra, S.

    1982-01-01

    Vertical slab gel electrophoresis of DNA with CH/sub 3/HgOH-containing agarose produces sharp bands whose mobilities are suitable for size estimation of single-stranded DNA containing 600 to 20,000 bases. The relationship of electrophoretic mobility to size of DNA over this range is a smooth, S-shaped function, and an empirical model was developed to express the relationship. The model involves terms in squared and reciprocal mobilities, and produced excellent fit of known standard markers to measured mobilities. It was used to estimate the sizes of six parvovirus DNAs: Kilham rat virus (KRV), H-1, LuIII, and minute virus of mice (MVM) DNAs had molecular weights of 1.66 to 1.70 x 10/sup 6/, while the molecular weight of bovine parvovirus (BPV) DNA was 1.84 x 10/sup 6/ and that of adenoassociated virus (AAV) DNA was 1.52 x 10/sup 6/.

  18. Polysaccharides of algae. Pt. 37. Characterization of hybrid structure of substituted agarose from Polysiphonia morrowii (Rhodophyta, Rhodomelaceae) using. beta. -agarase and /sup 13/C-NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Usov, A.I.; Ivanova, E.G.

    1987-09-01

    Structure of gel-forming galactan from Polysiphonia morrowii was analysed using bacterial ..beta..-agarase and /sup 13/C-nuclear magnetic resonance (/sup 13/C-NMR) spectroscopy. The polysaccharide was shown to contain: a) blocks composed of agarobiose residues, partly 6-O-methylated and 6-sulfated, which are sensitive to enzymolysis; b) extended blocks composed of agarobiose 6-sulfate residues, which are resistant to ..beta..-agarase action. The latter blocks contain also ..beta..-D-galactopyranosyl-(1->4)-..cap alpha..-L-galactopyranose 6.6'-disulfate residues (biogenetic precursors of agarobiose 6-sulfate), which are hardly detectable by /sup 13/C-NMR spectrum of the starting polysaccharide. Action of alkali on the enzyme-resistant fraction afforded a polysaccharide preparation having /sup 13/C-NMR spectrum of agarose 6-sulfate.

  19. Comparison of the capillary and agarose electrophoresis based multiple locus VNTR (variable number of tandem repeats) analysis (MLVA) on Mycobacterium bovis isolates.

    Science.gov (United States)

    Jenkins, A O; Venter, E H; Hutamo, K; Godfroid, J

    2010-09-28

    Electrophoretic techniques that can be used for genotyping of bacterial pathogens ranges from manual, low-cost, agarose gels to high-throughput capillary electrophoresis sequencing machines. These two methods are currently employed in the electrophoresis of PCR products used in multiple locus VNTR (variable number of tandem repeats) analysis (MLVA), i.e. the agarose electrophoresis (AE) and the capillary electrophoresis (CE). Some authors have suggested that clusters generated by AE are less reliable than those generated by CE and that the latter is a more sensitive technique than the former when typing Mycobacterium tuberculosis complex (MTC) isolates. Because such a claim could have significant consequences for investigators in this field, a comparison was made on 19 Belgian Mycobacterium bovis strains which had previously been genotyped using CE VNTR analysis. The VNTR profiles of the CE VNTR analysis were compared with those obtained by AE VNTR analysis at 14 VNTR loci. Our results indicated that there were no differences in copy numbers at all loci tested when the copy numbers obtained by the AE VNTR analysis were compared with those obtained by CE VNTR analysis. The use of AE VNTR analysis in mycobacterial genotyping does not alter the sensitivity of the MLVA technique compared with the CE VNTR analysis. The AE VNTR can therefore be regarded as a viable alternative in moderately equipped laboratories that cannot afford the expensive equipment required for CE VNTR analysis and data obtained by AE VNTR analysis can be shared between laboratories which use the CE VNTR method. (c) 2010 Elsevier B.V. All rights reserved.

  20. Immobilization/Stabilization of Ficin Extract on Glutaraldehyde-Activated Agarose Beads. Variables That Control the Final Stability and Activity in Protein Hydrolyses

    Directory of Open Access Journals (Sweden)

    El-Hocine Siar

    2018-04-01

    Full Text Available Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated. At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

  1. Functionalized agarose as an effective and novel matrix for immobilizing Cicer arietinum β-galactosidase and its application in lactose hydrolysis

    Directory of Open Access Journals (Sweden)

    Rukhsana Satar

    Full Text Available Abstract The present study demonstrates the immobilization of β-galactosidase from Cicer arietinum on a simple and inexpensive matrix, glutaraldehyde functionalized agarose (GFA, to suggest its potential application in hydrolyzing whey lactose in biotechnology industries. The designed matrix provided large surface area for the immobilization of β-galactosidase, apart from exhibiting greater biocatalytic activity in terms of selectivity, loading and stability. GFA retained 83% enzyme activity as a result of immobilization. Soluble and GFA bound Cicer arietinum β-galactosidase showed the same pH and temperature-optima at pH 5.0 and at 50 °C, respectively. However, immobilized enzyme exhibited a greater fraction of activity at both acidic and basic pH, and at higher temperature ranges. GFA bound enzyme lost only 20 % enzyme in the presence of 3% galactose, and retained 70 % activity even after its sixth repeated use. Immobilized enzyme showed pronounced lactose hydrolysis from whey in batch processes at 55 °C as compared to enzyme in solution.

  2. Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites

    Directory of Open Access Journals (Sweden)

    Jose J. Virgen-Ortíz

    2017-01-01

    Full Text Available Lipases from Candida antarctica (isoform B and Rhizomucor miehei (CALB and RML have been immobilized on octyl-agarose (OC and further coated with polyethylenimine (PEI and dextran sulfate (DS. The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed. The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.

  3. Highly selective and sensitive optical sensor for determination of Pb2+and Hg2+ ions based on the covalent immobilization of dithizone on agarose membrane

    Science.gov (United States)

    Zargoosh, Kiomars; Babadi, Fatemeh Farhadian

    2015-02-01

    A highly sensitive and selective optical membrane for determination of Hg2+ and Pb2+ was prepared by covalent immobilization of dithizone on agarose membrane. In addition to its high stability, reproducibility and relatively long lifetime, the proposed optical sensor revealed good selectivity for target ions over a large number of alkali, alkaline earth, transition, and heavy metal ions. The proposed optical membrane displays linear responses from 1.1 × 10-8 to 2.0 × 10-6 mol L-1 and 1.2 × 10-8 to 2.4 × 10-6 mol L-1 for Hg2+ and Pb2+, respectively. The limits of detection (LOD) were 2.0 × 10-9 mol L-1 and 4.0 × 10-9 mol L-1 for Hg2+ and Pb2, respectively. The prepared optical membrane was successfully applied to the determination of Hg2+ and Pb2+ in industrial wastes, spiked tap water and natural waters without any preconcentration step.

  4. Temporal effect of inertial cavitation with and without microbubbles on surface deformation of agarose S gel in the presence of 1-MHz focused ultrasound.

    Science.gov (United States)

    Tomita, Y; Matsuura, T; Kodama, T

    2015-01-01

    Sonoporation has the potential to deliver extraneous molecules into a target tissue non-invasively. There have been numerous investigations of cell membrane permeabilization induced by microbubbles, but very few studies have been carried out to investigate sonoporation by inertial cavitation, especially from a temporal perspective. In the present paper, we show the temporal variations in nano/micro-pit formations following the collapse of inertial cavitation bubbles, with and without Sonazoid® microbubbles. Using agarose S gel as a target material, erosion experiments were conducted in the presence of 1-MHz focused ultrasound applied for various exposure times, Tex (0.002-60 s). Conventional microscopy was used to measure temporal variations in micrometer-scale pit numbers, and atomic force microscopy utilized to detect surface roughness on a nanometer scale. The results demonstrated that nanometer-scale erosion was predominantly caused by Sonazoid® microbubbles and C4F10 gas bubbles for 0.002 scavitation bubbles such as C4F10 gas bubbles and vapor bubbles, increased exponentially with increasing Tex in the range 0.1 scavitation-induced sonoporation can produce various pore sizes in membranes, enabling the delivery of external molecules of differing sizes into cells or tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    Science.gov (United States)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  6. A novel homocystine-agarose adsorbent for separation and preconcentration of nickel in table salt and baking soda using factorial design optimization of the experimental conditions.

    Science.gov (United States)

    Hashemi, Payman; Rahmani, Zohreh

    2006-02-28

    Homocystine was for the first time, chemically linked to a highly cross-linked agarose support (Novarose) to be employed as a chelating adsorbent for preconcentration and AAS determination of nickel in table salt and baking soda. Nickel is quantitatively adsorbed on a small column packed with 0.25ml of the adsorbent, in a pH range of 5.5-6.5 and simply eluted with 5ml of a 1moll(-1) hydrochloric acid solution. A factorial design was used for optimization of the effects of five different variables on the recovery of nickel. The results indicated that the factors of flow rate and column length, and the interactions between pH and sample volume are significant. In the optimized conditions, the column could tolerate salt concentrations up to 0.5moll(-1) and sample volumes beyond 500ml. Matrix ions of Mg(2+) and Ca(2+), with a concentration of 200mgl(-1), and potentially interfering ions of Cd(2+), Cu(2+), Zn(2+) and Mn(2+), with a concentration of 10mgl(-1), did not have significant effect on the analyte's signal. Preconcentration factors up to 100 and a detection limit of 0.49mugl(-1), corresponding to an enrichment volume of 500ml, were obtained for the determination of the analyte by flame AAS. Application of the method to the determination of natural and spiked nickel in table salt and baking soda solutions resulted in quantitative recoveries. Direct ETAAS determination of nickel in the same samples was not possible because of a high background observed.

  7. High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs.

    Science.gov (United States)

    Giori, Luca; Tricomi, Flavia Marcella; Zatelli, Andrea; Roura, Xavier; Paltrinieri, Saverio

    2011-07-01

    The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: 0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

  8. Coeficientes de difusão de metais em materiais não convencionais (agarose e acetato de celulose usados na técnica de difusão em filmes finos por gradientes de concentração

    Directory of Open Access Journals (Sweden)

    Camila Destro Colaço

    2012-01-01

    Full Text Available The DGT technique allows one to measure quantitatively free and labile metal species in aquatic systems. Nevertheless, for this approach, knowledge is required of the diffusion coefficients of the analytes in a diffusive layer. In this study, the diffusion coefficients of Hg(II, As(III, Mn(II, Mg(II, Cu(II, Cd(II were determined in agarose gel and those of Ba(II, Cd(II, Cu(II, Mg(II, Mn(II e Zn(II in cellulose acetate membranes. These materials presented good performance and the reported results can be used as a data base for further DGT studies.

  9. A lightweight scalable agarose-gel-synthesized thermoelectric composite

    Science.gov (United States)

    Kim, Jin Ho; Fernandes, Gustavo E.; Lee, Do-Joong; Hirst, Elizabeth S.; Osgood, Richard M., III; Xu, Jimmy

    2018-03-01

    Electronic devices are now advancing beyond classical, rigid systems and moving into lighweight flexible regimes, enabling new applications such as body-wearables and ‘e-textiles’. To support this new electronic platform, composite materials that are highly conductive yet scalable, flexible, and wearable are needed. Materials with high electrical conductivity often have poor thermoelectric properties because their thermal transport is made greater by the same factors as their electronic conductivity. We demonstrate, in proof-of-principle experiments, that a novel binary composite can disrupt thermal (phononic) transport, while maintaining high electrical conductivity, thus yielding promising thermoelectric properties. Highly conductive Multi-Wall Carbon Nanotube (MWCNT) composites are combined with a low-band gap semiconductor, PbS. The work functions of the two materials are closely matched, minimizing the electrical contact resistance within the composite. Disparities in the speed of sound in MWCNTs and PbS help to inhibit phonon propagation, and boundary layer scattering at interfaces between these two materials lead to large Seebeck coefficient (> 150 μV/K) (Mott N F and Davis E A 1971 Electronic Processes in Non-crystalline Materials (Oxford: Clarendon), p 47) and a power factor as high as 10 μW/(K2 m). The overall fabrication process is not only scalable but also conformal and compatible with large-area flexible hosts including metal sheets, films, coatings, possibly arrays of fibers, textiles and fabrics. We explain the behavior of this novel thermoelectric material platform in terms of differing length scales for electrical conductivity and phononic heat transfer, and explore new material configurations for potentially lightweight and flexible thermoelectric devices that could be networked in a textile.

  10. Recovery of DNA from agarose gel by trap method

    African Journals Online (AJOL)

    Administrator

    2011-09-05

    Sep 5, 2011 ... gels, which can recover DNA with common laboratory facilities. This way can provide another ... which is difficult for the micro centrifuge. But our DNA could be extracted from n-butanol. ... taken out from the traps to 15 ml centrifuge tubes with a Pasteur pipette. The volume of the buffer extracted was equal to ...

  11. Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

    Science.gov (United States)

    Rio, Donald C

    2015-03-02

    In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

  12. Optical properties of an anterior lamellar human cornea model based on fibrin-agarose

    Science.gov (United States)

    Ionescu, Ana M.; Cardona, Juan de la Cruz; Ghinea, Razvan; Garzón, Ingrid; González-Andrades, Miguel; Alaminos, Miguel; Pérez, Maria del Mar

    2017-08-01

    The optical evaluation carried out using the Inverse Adding-Doubling (IAD) method to determine the scattering and the absorption coefficients of the bioengineered human corneal stromas showed that this type of artificial biomaterials shared many similarities with native control cornea after four weeks of development in culture. Their absorption and reduced scattering coefficients values were higher than the ones of the control cornea, but their spectral behaviors of both coefficients were similar. Time of development in culture was an influencing factor on the results.

  13. Analyzing modifiers of protein aggregation in C. elegans by native agarose gel electrophoresis

    NARCIS (Netherlands)

    Holmberg, Mats; Nollen, Ellen A A; Hatters, Danny M.; Hannan, Anthony J.

    2013-01-01

    The accumulation of specific aggregation-prone proteins during aging is thought to be involved in several diseases, most notably Alzheimer's and Parkinson's disease as well as polyglutamine expansion disorders such as Huntington's disease. Caenorhabditis elegans disease models with transgenic

  14. Assessing the Efficacy of First-Aid Measures in Physalia sp. Envenomation, Using Solution- and Blood Agarose-Based Models

    Directory of Open Access Journals (Sweden)

    Christie L. Wilcox

    2017-04-01

    Full Text Available Stings from the hydrozoan species in the genus Physalia cause intense, immediate skin pain and elicit serious systemic effects. There has been much scientific debate about the most appropriate first aid for these stings, particularly with regard to whether vinegar use is appropriate (most current recommendations recommend against vinegar. We found that only a small percentage (≤1.0% of tentacle cnidae discharge during a sting event using an ex vivo tissue model which elicits spontaneous stinging from live cnidarian tentacles. We then tested a variety of rinse solutions on both Atlantic and Pacific Physalia species to determine if they elicit cnidae discharge, further investigating any that did not cause immediate significant discharge to determine if they are able to inhibit cnidae discharge in response to chemical and physical stimuli. We found commercially available vinegars, as well as the recently developed Sting No More® Spray, were the most effective rinse solutions, as they irreversibly inhibited cnidae discharge. However, even slight dilution of vinegar reduced its protective effects. Alcohols and folk remedies, such as urine, baking soda and shaving cream, caused varying amounts of immediate cnidae discharge and failed to inhibit further discharge, and thus likely worsen stings.

  15. Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties.

    Science.gov (United States)

    Zsolnai, A; Orbán, L; Chrambach, A

    1993-03-01

    Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel ("sea-level electrophoresis"), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA system, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer.

  16. Multilocus enzyme electrophoresis on agarose gel as an aid to the identification of entomopathogenic Bacillus sphaericus strains.

    Science.gov (United States)

    Zahner, V; Rabinovitch, L; Cavados, C F; Momen, H

    1994-04-01

    Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types). Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype. Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40). The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C. 3.4.13.9) were polymorphic. The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group. This latter group appears to be distinct from other strains of these species. All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

  17. Assessing the Efficacy of First-Aid Measures in Physalia sp. Envenomation, Using Solution- and Blood Agarose-Based Models

    Science.gov (United States)

    Wilcox, Christie L.; Headlam, Jasmine L.; Doyle, Thomas K.; Yanagihara, Angel A.

    2017-01-01

    Stings from the hydrozoan species in the genus Physalia cause intense, immediate skin pain and elicit serious systemic effects. There has been much scientific debate about the most appropriate first aid for these stings, particularly with regard to whether vinegar use is appropriate (most current recommendations recommend against vinegar). We found that only a small percentage (≤1.0%) of tentacle cnidae discharge during a sting event using an ex vivo tissue model which elicits spontaneous stinging from live cnidarian tentacles. We then tested a variety of rinse solutions on both Atlantic and Pacific Physalia species to determine if they elicit cnidae discharge, further investigating any that did not cause immediate significant discharge to determine if they are able to inhibit cnidae discharge in response to chemical and physical stimuli. We found commercially available vinegars, as well as the recently developed Sting No More® Spray, were the most effective rinse solutions, as they irreversibly inhibited cnidae discharge. However, even slight dilution of vinegar reduced its protective effects. Alcohols and folk remedies, such as urine, baking soda and shaving cream, caused varying amounts of immediate cnidae discharge and failed to inhibit further discharge, and thus likely worsen stings. PMID:28445412

  18. Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture.

    Science.gov (United States)

    Guo, Xiaoling; Li, Shanyi; Ji, Qingshan; Lian, Ruiling; Chen, Jiansu

    2015-10-01

    Human adipose-derived stem cells (hADSCs) are potential adult stem cells source for cell therapy. But hADSCs with multi-passage or cryopreservation often revealed poor growth performance. The aim of our work was to improve the activity of poor post-thaw hADSCs by simple and effective means. We describe here a simple method based on commercially available silicone micro-wells for creating hADSCs spheroids to improve viability and neural differentiation potential on poor post-thaw hADSCs. The isolated hADSCs positively expresse d CD29, CD44, CD105, and negatively expressed CD34, CD45, HLA-DR by flow cytometry. Meanwhile, they had adipogenic and osteogenic differentiation capacity. The post-thaw and post-spheroid hADSCs from poor growth status hADSCs showed a marked increase in cell proliferation by CKK-8 analysis, cell cycle analysis and Ki67/P27 quantitative polymerase chain reaction (qPCR) analysis. They also displayed an increase viability of anti-apoptosis by annexin v and propidium iodide assays and mitochondrial membrane potential assays. After 3 days of neural induction, the neural differentiation potential of post-thaw and post-spheroid hADSCs could be enhanced by qPCR analysis and western blotting analysis. These results suggested that the spheroid formation could improve the viability and neural differentiation potential of bad growth status hADSCs, which is conducive to ADSCs research and cell therapy.

  19. Results concerning the analysis of the reaction products resulting from genomic dna amplification using agarose gel electrophoresis for potatoes studied old varieties

    Directory of Open Access Journals (Sweden)

    Anca BACIU

    2008-05-01

    Full Text Available The author is currently involved in collecting, making an inventory, evaluation and preservation the old varieties from the Western part of Romania. In this paper 8 potato old varieties collected during 20 years and 2 varieties from National Institute of Research and Development for potato and Sugar Beet Brasov are presented. The preservation was carried out in vivo and in vitro. Important changes were observed during this time. In our work we identified many gaps in the knowledge and understanding of the origin of transformations. We made a comparison between two big areas of potato growth: Apuseni Mountains [5] and the Maramures County [3]. In these areas the potato represents the main food in winter. This work opens opportunities for future researches in the field of political and ethical decisions for potato gene pool conservation. Soon the exchange of genetic resources will be a diplomatic issue.

  20. Molecular study and phylogenetic analysis of Mycoplasma synoviae ...

    African Journals Online (AJOL)

    Dr.Hosseini

    2012-01-26

    Jan 26, 2012 ... 4Clinical Sciences Department, Faculty of Veterinary Science, University of Tehran, Iran. .... France) in 2% agarose (Agarose MP, Roche) gel in TAE buffer. ... were performed with sequencing project management and.

  1. Morphological Instabilities in a Growing Yeast Colony: Experiment and Theory

    DEFF Research Database (Denmark)

    Sams, Thomas; Sneppen, Kim; Jensen, Mogens

    1997-01-01

    We study the growth of colonies of the yeast Pichia membranaefaciens on agarose film. The growth conditions are controlled in a setup where nutrients are supplied through an agarose film suspended over a solution of nutrients. As the thickness of the agarose film is varied, the morphology of the ...

  2. The ferric aerobactin receptor IutA, a protein isolated on agarose column, is not essential for uropathogenic Escherichia coli infection El receptor de aerobactina IutA, una proteína aislada en columna de agarosa, no es esencial para la infección por Escherichia coli uropatógena O receptor de aerobactina IutA, uma proteína isolada em coluna de agarose, não é essencial para a infecção por Escherichia coli uropatogênica

    Directory of Open Access Journals (Sweden)

    Taise Natali Landgraf

    2012-04-01

    Full Text Available Although many proteins have been described involved in Escherichia coli colonization and infection, only few reports have shown lectins as important components in these processes. Because the mechanisms underlying E. coli colonization process involving lectins are not fully understood, we sought to identify the presence of other non-described lectins in E. coli. Here, we isolated a 75-kDa protein from E. coli on Sepharose column and identified it as ferric aerobactin receptor (IutA. Since IutA is controversially associated with virulence of some E. coli strains, mainly in uropathogenic E. coli (UPEC, we evaluated the presence of iutA gene in UPEC isolated from patients with urinary infection. This gene was present in only 38% of the isolates, suggesting a weak association with virulence. Because there is a redundancy in the siderophore-mediated uptake systems, we suggest that IutA can be advantageous but not essential for UPEC.La falta de una clara comprensión de los mecanismos de participación de las lectinas en el proceso de colonización por Escherichia coli, nos motivó a identificar la presencia de otras lectinas que no han sido descritas en E. coli. En este estudio, se aisló una proteína de 75kDa de E. coli en una columna de Sepharosa, correspondiente al receptor de aerobactina (IutA. La asociación de IutA con cepas virulentas de E coli es controvertido, especialmente en E. coli uropatógena (UPEC, lo que nos llevó a evaluar la presencia del gen iutA en UPECs aisladas de pacientes con infección urinaria. El gen estaba presente en 38% de los aislamientos, lo que sugiere una débil asociación con la virulencia. Debido a la existencia de redundancia en los sistemas de captura de hierro, se sugiere que IutA puede ser una ventaja, sin embargo no es esencial para la UPEC.Apenas alguns relatos na literatura demonstram que lectinas são importantes nos processos de colonização e infecção por Escherichia coli. A falta de compreensão clara dos mecanismos envolvendo lectinas, no processo de colonização por E. coli, motivou a realização deste estudo para se identificar a presença de outras lectinas não descritas em E. coli. Neste trabalho, isolou-se uma proteína de 75kDa de E. coli em coluna de Sepharose, correspondente ao receptor de aerobactina férrica (IutA. A associação de IutA com virulência de cepas de E. coli é controversa, principalmente em E. coli uropatogênica (UPEC, o que levou a se avaliar a presença do gene iutA em UPECs isoladas de pacientes com infecção urinária. O gene estava presente em 38% dos isolados, sugerindo fraca associação com virulência. Devido à existência de redundância nos sistemas de captura de ferro, sugere-se, aqui, que IutA possa ser vantajosa, mas não essencial para UPEC.

  3. Alternative Methods in Laboratory Diagnosis of Equine Piroplasmosis

    African Journals Online (AJOL)

    Similar observation was made when such DNA material was examined in 1.5% agarose gel electrophoresis. Polymerase chain reaction of these cultures and purified genomic DNA yielded 260 base pair fragments diagnostic of B. equi infection. Spectrophotometry and agarose gel electrophoresis only indicate presence of ...

  4. Optimization of microsatellite DNA Gelred fluorescence imaging ...

    African Journals Online (AJOL)

    user1

    2012-10-11

    Oct 11, 2012 ... In order to explore the best microsatellite DNA Gelred imaging technology, this ... analysis and character identification breeding practice, because it is ... detection methods are agarose gel electrophoresis (AGE) with ethidium ... method (PG). Gelred 10000X stock reagent was diluted in the 1.5% agarose gel.

  5. Polymers in cell encapsulation from an enveloped cell perspective

    NARCIS (Netherlands)

    de Vos, Paul; Lazarjani, Hamideh Aghajani; Poncelet, Denis; Faas, Marijke M.

    2014-01-01

    In the past two decades, many polymers have been proposed for producing immunoprotective capsules. Examples include the natural polymers alginate, agarose, chitosan, cellulose, collagen, and xanthan and synthetic polymers poly(ethylene glycol), polyvinyl alcohol, polyurethane, poly(ether-sulfone),

  6. Simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

    Energy Technology Data Exchange (ETDEWEB)

    Lemon, S M; Jansen, R W

    1985-06-01

    Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.

  7. An Efficient Covalent Coating on Glass Slides for Preparation of Optical Oligonucleotide Microarrays

    Directory of Open Access Journals (Sweden)

    Atefeh Pourjahed

    2013-12-01

    The agarose-PLL microarrays had the highest signal (2546 and lowest background signal (205 in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes.

  8. CHAPTER 1

    African Journals Online (AJOL)

    Dr Olaleye

    The amplicons were sequenced and subjected to basic local alignment search tool ... Out of the 12 amplicons in agarose gel, there were 6 sharp and 6 faint bands .... A: Consensus sequence with (-) and (*) signs representing gaps and points ...

  9. Expression of human interferon gamma in Brassica napus seeds

    African Journals Online (AJOL)

    TUOYO

    2010-08-09

    Aug 9, 2010 ... resulted band was purified using the agarose gel DNA extraction kit. (Roche). ..... rape seed napin structure and potential roles of the storage protein. ... the sensitivity of progressive multiple sequence alignment through.

  10. Fulltext PDF

    Indian Academy of Sciences (India)

    Wintec

    Synthesis and characterization of new meso- substituted ... properties of palladium: Metal nanoparticle and thin film over ... Agarose-metal/semiconductor nanoparticle films. Synthesis of ... Tuning of Ag-SPR band position in refractive index.

  11. Polymorphism of angiotensin converting enzyme (insertion/deletion ...

    Indian Academy of Sciences (India)

    cells: neuronal (nNOS, type I), inducible (iNOS, type II) and endothelial ... was detected on 2% agarose gel containing ethidium bro- mide. .... Gender, male N (%). 329 (40.5%) .... ischemic heart disease in Japanese diabetic subjects. Diabetes.

  12. Molecular cloning and characterization of the plasma membrane ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... Southern blot analysis indicated that OvPIP gene was present in O. ... Key words: Orychophragmus violaceus, plasma membrane, tonoplast aquaporins .... fractionated by 0.8% agarose gel electrophoresis and transferred to.

  13. Studies on plant regeneration and transformation efficiency of ...

    African Journals Online (AJOL)

    Jane

    2010-10-11

    Oct 11, 2010 ... most used method for the introduction of foreign genes into plant cells and the subsequent ... purine; NAA, α-naphthaleneacetic acid; MS, Murashige and. Skoog; nptII .... The amplified product was analyzed in 1% agarose.

  14. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    2-5A; antiviral function; cellular RNA degradation; interferons; recombinant RNase L ... yield and degradation of the recombinant protein, which demands number of ... A semi-quantitative agarose-gel-based ribonuclease assay was developed ...

  15. 16SrRNA and enzymatic diversity of culturable bacteria from the sediments of oxygen minimum zone in the Arabian Sea

    Digital Repository Service at National Institute of Oceanography (India)

    Divya, B.; Soumya, K.V.; Nair, S.

    hour. The restricted products were electrophoresed in 1.2% agarose gel and viewed on Gel Imaging System (Kodak Gel Logic). The patterns in the gels were compared using Bionumerics Software Version 4.6 (Applied Maths, Belgium). Representative...

  16. Biochemical and molecular characterizaion of Bacillus pumilus isolated from coastal environment in Cochin, India

    Digital Repository Service at National Institute of Oceanography (India)

    Parvathi, A.; Krishna, K.; Jose, J.; Joseph, N.; Nair, S.

    products were separated on a 2% agarose gel, stained with ethidium bromide and photographed. Amplification profiles obtained were analyzed and a dendrogram was generated using BioNumerics version 4.6 software (Applied Maths, Belgium) 7 PCR...

  17. Optimization of RAPD-PCR reaction system for genetic relationships ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-02-08

    Feb 8, 2010 ... Camellia features beautiful tree shape, various petal color, .... (Huzhuabai (C. japonica)) was randomly selected for optimization of .... Afr. J. Biotechnol. Figure 1. Agarose gel electrophoresis analysis by primer S256 (left-up), ...

  18. Potential of Raloxifene in reversing osteoarthritis-like alterations in ...

    Indian Academy of Sciences (India)

    2012-12-16

    Dec 16, 2012 ... 135. Keywords. Agarose; cartilage; chondroprotective; in vitro models; osteoarthritis; Raloxifene ...... could be attributed to variations in the organization of the .... O'Hara BP, Urban JP and Maroudas A 1990 Influence of cyclic.

  19. The Morse code effect: A crystal-crystal transformation observed in gel-grown lead (II) oxalate crystals

    Science.gov (United States)

    Lisgarten, J. N.; Marks, J. A.

    2018-05-01

    This paper reports on an unusual crystal-crystal transformation phenomenon, which we have called the Morse Code Effect, based on the change in appearance of lead(II) oxalate crystals grown in agarose gels.

  20. Non-stationary heat transfer in gels applied to biotehnology

    Directory of Open Access Journals (Sweden)

    Pokusaev Boris

    2017-01-01

    Full Text Available Unsteady heat transfer in agarose gels of various concentrations was studied in order to make a breakthrough in the technology of 3-D additive bioprinting. Data on the kinetics of the phase transformation was obtained using spectroscopy as a function of temperature during the formation of agarose hydrogel. The dynamics of aging was investigated for gels of different densities. The time dependence of the structural changes was obtained. Particular attention was paid to the changes in the structure of the gel due to the processes of evaporation of the liquid during the gel formation and during long-term storage. Experiments were performed to determine the dynamics of the temperature fields simultaneously with heat flux measurements during the formation of agarose gels from different initial concentrations. A technique based on experimental data for the computations of the thermophysical coefficients of agarose gels was developed.

  1. Studies on Agrobacterium-mediated genetic transformation of ...

    African Journals Online (AJOL)

    SERVER

    2008-03-04

    Mar 4, 2008 ... Sweet potato (Ipomoea batatas) is the sixth most impor- tant crop in the world after ... mediated transformation system does not involve sophis- .... (w/v) agarose gel. .... This work was supported by National Natural Science.

  2. 21 CFR 866.4900 - Support gel.

    Science.gov (United States)

    2010-04-01

    ... IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866.4900 Support gel. (a) Identification. A support gel for clinical use is a device that consists of an agar or agarose preparation that...

  3. A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

    International Nuclear Information System (INIS)

    Lemon, S.M.; Jansen, R.W.

    1985-01-01

    Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus. (Auth.)

  4. Fulltext PDF

    Indian Academy of Sciences (India)

    rants and in fish markets due to consumption of their eggs by the local people. ... The PCR products were separated by electrophoresis on 2%. TBE agarose gel and ... Carlsbad, USA) and sent to First Base Laboratory (Seri. Kembangan ...

  5. Construction and analysis of subtractive hybridization library of ...

    African Journals Online (AJOL)

    Yomi

    2012-03-22

    Mar 22, 2012 ... 1Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, .... Mix, Agarose Gel DNA purification kit, pMD®19-T Vector, DNA ...... the interface between membrane traffic and cell signalling. EMBO.

  6. Molecular characterization, expression profile of the FSHR gene and ...

    Indian Academy of Sciences (India)

    2016-10-24

    Oct 24, 2016 ... gene(internal control), respectively.P5~P13 ... The obtained RNA quality was detected by 1.5% agarose gel electrophoresis and the concentration was ... sequenced by a commercial service (Majorbio Co. Ltd, Shanghai).

  7. Download

    African Journals Online (AJOL)

    userpc

    to humans as a result of consumption of ... Genomic DNA isolation from Milk sample. (phenol ... amplification of all the extracted genomic DNA .... PLATE 1: A two panel agarose gel electrophoresis of PCR amplification of oxyR gene specific.

  8. Cloning and expression of porcine SRPK1 gene

    African Journals Online (AJOL)

    Academic Journals

    2012-01-10

    Jan 10, 2012 ... different porcine tissue and skeletal muscle repair processes. ... Biology Engineering Technology Service Co., Ltd; while ethanol, agarose gel DNA ..... muscle fiber regeneration after bupivacaine hydrochloride-and acid.

  9. Dosimetry Evolution in Teletherapy: Polimer Gel

    Science.gov (United States)

    Hamann, J. H.; Peixoto, J. G. P.

    2018-03-01

    Polymer gels evolution and chemical composition used in dosimetry. Type Composition First gels Folin’s Phenol or Gallic Acid Polymer Gel Agarose and N,N’-methylene-bis-acrylamide BANANA Bis, acrylamide, nitrous oxide and agarose BANG-1TM Bis, acrylamide, nitrogen and gelatin BANG-2TM Bis, acrylic acid, sodium hydroxide, nitrogen and gelatin BANG-3TM Bis, methacrylate acid, sodium hydroxide, nitrogen and gelatin MAGIC Methacrylate acid, ascorbic acid, gelatin and copper sulphate

  10. STEM VQ Method, Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification

    Science.gov (United States)

    2017-02-02

    WEEV in Ontario, Canada in 194126. This virus has a passage history including both animals and cell culture. Biosafety level (BSL-)-3 laboratory...Agarose-Based Plaque Assay Each virus stock was quantitated by standard agarose-based plaque assay23...samples used here were well prepared and the standard macro was used. As we have developed this method we have observed that while inferior

  11. Electrophoretic mobility of PM2 DNA treated with ultimate chemical carcinogens or with ultraviolet light

    International Nuclear Information System (INIS)

    Thielmann, H.W.; Hecht, R.

    1980-01-01

    Superhelical DNA of the Pseudomonas phage PM2 was irradiated with UV-light or reacted with covalently binding carcinogens, such as 7-bromomethyl-benz[a]anthracene, (Ac) 2 ONFln, K-region epoxides, and alkylating agents. Migration velocity of the DNA products was determined using agarose gel electrophoresis. In gels of more than 1.3%-1.9% agarose, modified PM2 DNA exhibited a dose-(concentration-)dependent decrease of migration velocity. This phenomenon is probably due to a decrease in superhelix density which caused the compact DNA coil to assume eventually an open-circular conformation. Comparison of the extent of DNA modification with the decrease of migration velocity revealed that the superhelical structure sensitively reflected the chemical DNA alterations. DNA species exhibiting in 1.6% agarose gels, a migration velocity of up to 30% of that of control DNA showed an increase of velocity in 0.4% agarose. Therefore, in 1.3%-1.9% agarose gels, the decrease of superhelix density is accompanied by an increase of the frictional coefficient, whereas in 0.4%-0.9% agarose gels the same decrease of superhelix density apparently led to a higher degree of flexibility of the macromolecule and/or exposure of additional electric charges. (orig.) [de

  12. Identification of adequate vehicles to carry nerve regeneration inducers using tubulisation.

    Science.gov (United States)

    do Nascimento-Elias, Adriana Helena; Fresnesdas, Bruno César; Schiavoni, Maria Cristina Lopes; de Almeida, Natália Fernanda Gaspar; Santos, Ana Paula; de Oliveira Ramos, Jean; Junior, Wilson Marques; Barreira, Amilton Antunes

    2012-08-14

    Axonal regeneration depends on many factors, such as the type of injury and repair, age, distance from the cell body and distance of the denervated muscle, loss of surrounding tissue and the type of injured nerve. Experimental models use tubulisation with a silicone tube to research regenerative factors and substances to induce regeneration. Agarose, collagen and DMEM (Dulbecco's modified Eagle's medium) can be used as vehicles. In this study, we compared the ability of these vehicles to induce rat sciatic nerve regeneration with the intent of finding the least active or inert substance. The experiment used 47 female Wistar rats, which were divided into four experimental groups (agarose 4%, agarose 0.4%, collagen, DMEM) and one normal control group. The right sciatic nerve was exposed, and an incision was made that created a 10 mm gap between the distal and proximal stumps. A silicone tube was grafted onto each stump, and the tubes were filled with the respective media. After 70 days, the sciatic nerve was removed. We evaluated the formation of a regeneration cable, nerve fibre growth, and the functional viability of the regenerated fibres. Comparison among the three vehicles showed that 0.4% agarose gels had almost no effect on provoking the regeneration of peripheral nerves and that 4% agarose gels completely prevented fibre growth. The others substances were associated with profuse nerve fibre growth. In the appropriate concentration, agarose gel may be an important vehicle for testing factors that induce regeneration without interfering with nerve growth.

  13. Biomimetic brain tumor niche regulates glioblastoma cells towards a cancer stem cell phenotype.

    Science.gov (United States)

    Liu, Yung-Chiang; Lee, I-Chi; Chen, Pin-Yuan

    2018-05-01

    Glioblastoma (GBM) is the most malignant primary brain tumor and contains tumorigenic cancer stem cells (CSCs), which support the progression of tumor growth. The selection of CSCs and facilitation of the brain tumor niches may assist the development of novel therapeutics for GBM. Herein, hydrogel materials composed of agarose and hydroxypropyl methyl cellulose (HMC) in different concentrations were established and compared to emulate brain tumor niches and CSC microenvironments within a label-free system. Human GBM cell line, U-87 MG, was cultured on a series of HMC-agarose based culture system. Cell aggregation and spheroids formation were investigated after 4 days of culture, and 2.5% HMC-agarose based culture system demonstrated the largest spheroids number and size. Moreover, CD133 marker expression of GBM cells after 6 days of culture in 2.5% HMC-agarose based culture system was 60%, relatively higher than the control group at only 15%. Additionally, cells on 2.5% HMC-agarose based culture system show the highest chemoresistance, even at the high dose of 500 µM temozolomide for 72 h, the live cell ratio was still > 80%. Furthermore, the results also indicate that the expression of ABCG2 gene was up-regulated after culture in 2.5% HMC-agarose based culture system. Therefore, our results demonstrated that biomimetic brain tumor microenvironment may regulate GBM cells towards the CSC phenotype and expression of CSC characteristics. The microenvironment selection and spheroids formation in HMC-agarose based culture system may provide a label-free CSC selection strategy and drug testing model for future biomedical applications.

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

    Science.gov (United States)

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

    2017-10-01

    Iron overload disorders may be treated by chelation therapy. This study describes a novel method for isolating iron chelators from complex mixtures including plant extracts. We demonstrate the one-step isolation of curcuminoids from turmeric, the medicinal food spice derived from Curcuma longa. The method uses iron-nitrilotriacetic acid (NTA)-agarose, to which curcumin binds rapidly, specifically, and reversibly. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin each bound iron-NTA-agarose with comparable affinities and a stoichiometry near 1. Analyses of binding efficiencies and purity demonstrated that curcuminoids comprise the primary iron binding compounds recovered from a crude turmeric extract. Competition of curcuminoid binding to the iron resin was used to characterize the metal binding site on curcumin and to detect iron binding by added chelators. Curcumin-Iron-NTA-agarose binding was inhibited by other metals with relative potency: (>90% inhibition) Cu 2+  ~ Al 3+  > Zn 2+  ≥ Ca 2+  ~ Mg 2+  ~ Mn 2+ (80% by addition of iron to the media; uptake was completely restored by desferoxamine. Ranking of metals by relative potencies for blocking curcumin uptake agreed with their relative potencies in blocking curcumin binding to iron-NTA-agarose. We conclude that curcumin can selectively bind toxic metals including iron in a physiological setting, and propose inhibition of curcumin binding to iron-NTA-agarose for iron chelator screening.

  15. Approach for growth of high-quality and large protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroyoshi, E-mail: matsumura@chem.eng.osaka-u.ac.jp [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Sugiyama, Shigeru; Hirose, Mika; Kakinouchi, Keisuke; Maruyama, Mihoko; Murai, Ryota [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); Adachi, Hiroaki; Takano, Kazufumi [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Murakami, Satoshi [JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Mori, Yusuke; Inoue, Tsuyoshi [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan)

    2011-01-01

    Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed. Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein–precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.

  16. Current knowledge on agarolytic enzymes and the industrial potential of agar-derived sugars.

    Science.gov (United States)

    Yun, Eun Ju; Yu, Sora; Kim, Kyoung Heon

    2017-07-01

    Agar is a major cell wall carbohydrate of red macroalgae (Rhodophyta). Sugars derived from agar, such as agarooligosaccharides (AOSs), neoagarooligosaccharides (NAOSs), neoagarobiose (NAB), and 3,6-anhydro-L-galactose (L-AHG), possess various physiological activities. These agar-derived sugars can be produced by hydrolysis using chemicals or agarolytic enzymes. Despite the industrial potential of agar-derived sugars, their application has been hampered mainly due to the absence of efficient processes for the liquefaction and saccharification of agar. In this review, we have focused on strategies for producing high value-added sugars from agarose via chemical or enzymatic liquefaction and enzymatic saccharification. The liquefaction of agarose is a key step for preventing gelling and increasing the solubility of agarose in water by prehydrolyzing agarose into AOSs or NAOSs. For the industrial use of agar-derived sugars, AOS, NAOS, NAB, and L-AHG can be used as functional biomaterials owing to their physiological activities such as antiinflammation, skin whitening, and moisturizing. Recently, it was reported that AHG could be considered as a new anticariogenic sugar to replace xylitol. This review provides a comprehensive overview of processes for the hydrolysis of agar or agarose to produce high value-added sugars and the industrial application of these sugars.

  17. Probing the Effects of Templating on the UV and Visible Light Photocatalytic Activity of Porous Nitrogen-Modified Titania Monoliths for Dye Removal.

    Science.gov (United States)

    Nursam, Natalita M; Wang, Xingdong; Tan, Jeannie Z Y; Caruso, Rachel A

    2016-07-13

    Porous nitrogen-modified titania (N-titania) monoliths with tailored morphologies were prepared using phase separation and agarose gel templating techniques. The doping and templating process were simultaneously carried out in a one-pot step using alcohol amine-assisted sol-gel chemistry. The amount of polymer used in the monoliths that were prepared using phase separation was shown to affect both the physical and optical properties: higher poly(ethylene glycol) content increased the specific surface area, porosity, and visible light absorption of the final materials. For the agarose-templated monoliths, the infiltration conditions affected the monolith morphology. A porous monolith with high surface area and the least shrinkage was obtained when the N containing alkoxide precursor was infiltrated into the agarose scaffolds at 60 °C. The effect of the diverse porous morphologies on the photocatalytic activity of N-titania was studied for the decomposition of methylene blue (MB) under visible and UV light irradiation. The highest visible light activity was achieved by the agarose-templated N-titania monolith, in part due to higher N incorporation. This sample also showed better UV activity, partly because of the higher specific surface area (up to 112 m(2) g(-1)) compared to the phase separation-induced monoliths (up to 103 m(2) g(-1)). Overall, agarose-templated, porous N-titania monoliths provided better features for effectively removing the MB contaminant.

  18. Fabrication of Sericin/Agrose Gel Loaded Lysozyme and Its Potential in Wound Dressing Application

    Directory of Open Access Journals (Sweden)

    Meirong Yang

    2018-04-01

    Full Text Available Sericin is a biomaterial resource for its significant biodegradability, biocompatibility, hydrophilicity, and reactivity. Designing a material with superabsorbent, antiseptic, and non-cytotoxic wound dressing properties is advantageous to reduce wound infection and promote wound healing. Herein, we propose an environment-friendly strategy to obtain an interpenetrating polymer network gel through blending sericin and agarose and freeze-drying. The physicochemical characterizations of the sericin/agarose gel including morphology, porosity, swelling behavior, crystallinity, secondary structure, and thermal property were well characterized. Subsequently, the lysozyme loaded sericin/agarose composite gel was successfully prepared by the solution impregnation method. To evaluate the potential of the lysozyme loaded sericin/agarose gel in wound dressing application, we analyzed the lysozyme loading and release, antimicrobial activity, and cytocompatibility of the resulting gel. The results showed the lysozyme loaded composite gel had high porosity, excellent water absorption property, and good antimicrobial activities against Escherichia coli and Staphylococcus aureus. Also, the lysozyme loaded gel showed excellent cytocompatibility on NIH3T3 and HEK293 cells. So, the lysozyme loaded sericin/agarose gel is a potential alternative biomaterial for wound dressing.

  19. Hyper alginate gel microbead formation by molecular diffusion at the hydrogel/droplet interface.

    Science.gov (United States)

    Hirama, Hirotada; Kambe, Taisuke; Aketagawa, Kyouhei; Ota, Taku; Moriguchi, Hiroyuki; Torii, Toru

    2013-01-15

    We report a simple method for forming monodispersed, uniformly shaped gel microbeads with precisely controlled sizes. The basis of our method is the placement of monodispersed sodium alginate droplets, formed by a microfluidic device, on an agarose slab gel containing a high-osmotic-pressure gelation agent (CaCl(2) aq.): (1) the droplets are cross-linked (gelated) due to the diffusion of the gelation agent from the agarose slab gel to the sodium alginate droplets and (2) the droplets simultaneously shrink to a fraction of their original size (slab gel. We verified the mass transfer mechanism between the droplet and the agarose slab gel. This method circumvents the limitations of gel microbead formation, such as the need to prepare microchannels of various sizes, microchannel clogging, and the deformation of the produced gel microbeads.

  20. Structure of gels layers with cells

    Science.gov (United States)

    Pokusaev, B. G.; Karlov, S. P.; Vyazmin, A. V.; Nekrasov, D. A.; Zakharov, N. S.; Khramtsov, D. P.; Skladnev, D. A.; Tyupa, D. V.

    2017-11-01

    The structure of two-layer agarose gels containing yeast cells is investigated experimentally by spectrometry, to shed a light on the theoretical foundations for the development of bioreactors by the method of 3D bioprinting. Due to division, cells overcome the layer of the dispersion phase separating successively applied layers of the agarose gel. However a gel layer of 100 μm thick with a high concentration of silver nanoparticles completely excludes the infiltration of yeast cells through it. A special sort of agarose is suggested where the concentration of silver nanoparticles formed by cells from salt of silver can serve as an indicator of the state of the yeast cells in the volume of the gel.

  1. Identification of adequate vehicles to carry nerve regeneration inducers using tubulisation

    Directory of Open Access Journals (Sweden)

    do Nascimento-Elias Adriana Helena

    2012-08-01

    Full Text Available Abstract Background Axonal regeneration depends on many factors, such as the type of injury and repair, age, distance from the cell body and distance of the denervated muscle, loss of surrounding tissue and the type of injured nerve. Experimental models use tubulisation with a silicone tube to research regenerative factors and substances to induce regeneration. Agarose, collagen and DMEM (Dulbecco’s modified Eagle’s medium can be used as vehicles. In this study, we compared the ability of these vehicles to induce rat sciatic nerve regeneration with the intent of finding the least active or inert substance. The experiment used 47 female Wistar rats, which were divided into four experimental groups (agarose 4%, agarose 0.4%, collagen, DMEM and one normal control group. The right sciatic nerve was exposed, and an incision was made that created a 10 mm gap between the distal and proximal stumps. A silicone tube was grafted onto each stump, and the tubes were filled with the respective media. After 70 days, the sciatic nerve was removed. We evaluated the formation of a regeneration cable, nerve fibre growth, and the functional viability of the regenerated fibres. Results Comparison among the three vehicles showed that 0.4% agarose gels had almost no effect on provoking the regeneration of peripheral nerves and that 4% agarose gels completely prevented fibre growth. The others substances were associated with profuse nerve fibre growth. Conclusions In the appropriate concentration, agarose gel may be an important vehicle for testing factors that induce regeneration without interfering with nerve growth.

  2. Effect of low dose tritium on mouse lymphocyte DNA estimated by comet assay

    International Nuclear Information System (INIS)

    Ichimasa, Yusuke; Otsuka, Kensuke; Maruyama, Satoko; Tauchi, Hiroshi; Ichimasa, Michiko; Uda, Tatsuhiko

    2003-01-01

    This paper deals with low dose effect of HTO on mouse lymphocytes DNA (in vitro irradiation) estimated by the comet assay using ICR male mouse of 20 to 23 weeks old. Lymphocytes were isolated by centrifugation of whole blood sample on Ficoll-Paque solution and embedded in agarose gel just after mixed with HTO. After lymphocytes were exposed to 17-50 mGy of HTO, the agarose gel slides were washed to remove HTO and cell lysis treatment on the slides was conducted before electrophoresis. The individual comets on stained slides after electrophoresis were analyzed using imaging software. No significant DNA damages were observed. (author)

  3. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand.

    Science.gov (United States)

    Yasukawa, Takehiro; Reyes, Aurelio; Cluett, Tricia J; Yang, Ming-Yao; Bowmaker, Mark; Jacobs, Howard T; Holt, Ian J

    2006-11-15

    Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5' ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands to DNA starts at defined loci, the most prominent of which maps, in mammalian mtDNA, in the vicinity of the site known as the light-strand origin.

  4. Organic influences on inorganic patterns of diffusion-controlled precipitation in gels

    Science.gov (United States)

    Barge, Laura M.; Nealson, Kenneth H.; Petruska, John

    2010-06-01

    The well-known AgNO 3/K 2CrO 4 reaction-diffusion system produces periodic bands of silver chromate precipitate in gelatin, but only randomly oriented crystals in agarose gel. We show that comparable bands can be produced in agarose gel by adding small amounts of simple organic acids (e.g., acetic acid, N-acetyl glycine, and N-acetyl alanine) that suppress crystal growth and promote formation of rounded particles of precipitate. These results indicate that α-carboxyl groups of amino acids or short peptides in gelatin under mildly acidic conditions can induce periodic band patterns in diffusion-controlled silver chromate precipitates.

  5. Monitoring of biofilm growth using ATR-leaky mode spectroscopy

    International Nuclear Information System (INIS)

    Leitz, M.; Franke, H.; Grattan, K.T.V.; Tamachkiarow, A.

    2002-01-01

    An approach to the in situ monitoring of biofilm formation using the technique of ATR-leaky mode spectroscopy is given as an example for the case of Cytophaga. The biofilm growth was studied on an aluminium layer and on a bilayer of the hydrogel agarose on aluminium. This metal was chosen because of its chemical stability in aqueous systems. The spectra obtained have been recorded using a flow cell to contain the suspension and nutrients over a period of several days. In the case considered using a prism surface coated with agarose, the experiments were performed by breeding in an incubator. (author)

  6. Taxonomy of economic seaweeds : with reference to some Pacific and Caribbean species

    OpenAIRE

    Abbott, Isabella A; Norris, James N

    1985-01-01

    The value of any seaweed crop is enhanced by the name under which the seaweed is sold, for the kind and quality of the seaweed product is announced with its name. Thus, though chemists may say that the agar from Gelidium species is the same as that from Gracilaria species, industry will pay more for Gelidium than for Gracilaria. (It might be so because the agarose fraction is higher in Gelidium, and agarose commands a higher price on its own.) In the case of the seaweeds that produce the coll...

  7. ヒト血清M蛋白のIgGH鎖サブクラスの型判定について -高分解能アガロースゲル等電点電気泳動法の分離と検出のための条件設定-

    OpenAIRE

    一村, 光子; 唐下, 博子; 崎山, 順子; 中田, 安成; 遠藤, 浩

    1994-01-01

    IgG Heavy-Chain subclass typing of human serum M-protein were isoelectrofocussed (IEF) in agarose gel, and then the bands of IgG were detected with peroxidase-conjugated anti-human IgG1-4 monoclonal antibodies on the same isoelectrofocussed agarose gel plate. This IEF enzyme immunodetection patterns were composed of four to eight discrete bands (The range of pI was 6.0 to 9.0). These bands were dependent on immunofixationbands. It was very specific and clear to detect the subclass of IgG Type...

  8. LP (a) levels and apo (a) phenotypes in urban black South African ...

    African Journals Online (AJOL)

    300 U/I), intermediate (300 - 700 UII) and high (> 700 UII) plasma Lp (a) ... Tygerberg Hospital, Tygerberg, W Cape ... up to 200-fold difference in Lp (a) concentrations.' In addition, ethnic .... An interesting observation was the large number of phenotypes, Le. ... sample of black Americans with a high-resolution SOS- agarose ...

  9. Cytotoxic activity and apoptotic induction of some edible Thai local ...

    African Journals Online (AJOL)

    inverted microscopy and DNA fragmentation using agarose gel electrophoresis. Results: P. ... However, further studies are needed to isolate the active compounds responsible for the cytotoxic ..... D-E: TL at 500 and 4,000 μg/mL. Arrows ...

  10. Actively Shaken In-Situ Passive Sampler Platform for Methylmercury and Organics

    Science.gov (United States)

    2016-02-01

    from a risk standpoint, methylmercury (MeHg), and to identify a polymer partitioning approach by developing and testing a range of polymeric ...an assortment of thiolated polymers for use in pharmaceuticals, where their ability to form sulfide bonds confers mucoadhesive properties that...agarose. The thiolated polymers tested in this set leached sulfur into solution, causing analytical interferences and confounding the results. For

  11. Identification of six potato virus Y isolates from Saudi Arabia

    African Journals Online (AJOL)

    Sherif

    2012-05-22

    May 22, 2012 ... PAGE) indicated the 34 kDa viral coat protein and agarose gel of the immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) products indicated the 800 bp cp gene. The sequences were aligned together, narrowed to six (one PVY-N and five PVY-O isolates) and then aligned.

  12. The Influences of LuxX in "Escherichia Coli" Biofilm Formation and Improving Teacher Quality through the Bio-Bus Program

    Science.gov (United States)

    Robbins, Chandan Morris

    2012-01-01

    The objectives of this work are: (1) to agarose-stabilize fragile biofilms for quantitative structure analysis; (2) to understand the influences of LuxS on biofilm formation; (3) to improve teacher quality by preparing Georgia's middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative…

  13. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  14. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    Science.gov (United States)

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels.…

  15. Affinity chromatography of serine proteases on the triazine dye ligand Cibacron Blue F3G-A

    DEFF Research Database (Denmark)

    Koch, C; Borg, L; Skjødt, K

    1998-01-01

    The interaction between complement component factor B and the triazine dye ligand Cibacron Blue F3G-A coupled to a cross-linked agarose matrix (Blue Sepharose) was found to involve the Bb part of the molecule, and to be inhibited by benzamidine. Human, chicken and rainbow trout factor B which had...

  16. The assessment of natural scaffolds ability in chondrogenic ...

    African Journals Online (AJOL)

    Arun Kumar Agnihotri

    1Stem Cell Laboratory, The Academic Center for Education, Culture and Research, ... between alginate and agarose groups in maintaining cells viable but, about chondrogenic .... Absorbance at 570 nm was measured on a ... as small cells with little cytoplasm and elliptic ... high density of cells and suitable interaction.

  17. Development of a gradient tube method for examining microbial ...

    African Journals Online (AJOL)

    2013-06-24

    Jun 24, 2013 ... as brackish and salt marshes and these biofilms may be an important component ... the bottom of a sterile 15 mℓ glass test tube, a 5 mℓ molten agarose plug ... B. Field-scale floating sulphur biofilm reactor developed to enable.

  18. High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

    NARCIS (Netherlands)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart A.; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

  19. Effects of Lymantria dispar feeding and mechanical wounding on ...

    African Journals Online (AJOL)

    Jane

    2011-07-18

    Jul 18, 2011 ... MATERIALS AND METHODS. Plants and L. dispar. The plants used were poplar (P. ... relative humidity and an average temperature of 24°C. Fully expanded leaves were treated by feeding and .... transition rate of 0.1°C s-1 to generate a melting curve. All samples were also electrophoresed in agarose ...

  20. Hybridization of plant virus ssRNAs Transferred to Hybond N membrane

    International Nuclear Information System (INIS)

    Kudela, O.; Kudelova, K.; Plaschke-Jakubik, K.

    1998-01-01

    In this paper we present a protocol for the non-denaturating agarose gel electrophoresis of plant virus ssRNAs, their blotting onto Hybond N membrane, and hybridization with [alpha 32 P]dNTP-labelled cDNA probe. The protocol is not pretentious on technical equipment, omits denaturation and neutralization steps and some chemical required in other modifications. (authors)

  1. Phase separation of in situ forming poly (lactide-co-glycolide acid) implants investigated using a hydrogel-based subcutaneous tissue surrogate and UV-vis imaging.

    Science.gov (United States)

    Sun, Yu; Jensen, Henrik; Petersen, Nickolaj J; Larsen, Susan W; Østergaard, Jesper

    2017-10-25

    Phase separation of in situ forming poly (lactide-co-glycolide acid) (PLGA) implants with agarose hydrogels as the provider of nonsolvent (water) mimicking subcutaneous tissue was investigated using a novel UV-vis imaging-based analytical platform. In situ forming implants of PLGA-1-methyl-2-pyrrolidinone and PLGA-triacetin representing fast and slow phase separating systems, respectively, were evaluated using this platform. Upon contact with the agarose hydrogel, the phase separation of the systems was followed by the study of changes in light transmission and absorbance as a function of time and position. For the PLGA-1-methyl-2-pyrrolidinone system, the rate of spatial phase separation was determined and found to decrease with increasing the PLGA concentration from 20% to 40% (w/w). Hydrogels with different agarose concentrations (1% and 10% (w/v)) were prepared for providing the nonsolvent, water, to the in situ forming PLGA implants simulating the injection site environment. The resulting implant morphology depended on the stiffness of hydrogel matrix, indicating that the matrix in which implants are formed is of importance. Overall, the work showed that the UV-vis imaging-based platform with an agarose hydrogel mimicking the subcutaneous tissue holds potential in providing bio-relevant and mechanistic information on the phase separation processes of in situ forming implants. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Constructing a proton titration curve from ion-step measurements, applied to a membrane with adsorbed protein

    NARCIS (Netherlands)

    Eijkel, Jan C.T.; Bosch, Coen; Olthuis, Wouter; Bergveld, Piet

    1997-01-01

    A new measuring method is described for obtaining a proton titration curve. The curve is obtained from a microporous composite membrane, consisting of polystyrene beads in an agarose matrix, with lysozyme molecules adsorbed to the bead surface. The membrane is incorporated into a sensor system by

  3. Genetic diversity of populations of Butia capitata endangered ...

    African Journals Online (AJOL)

    Hélida

    2015-03-18

    Mar 18, 2015 ... (polymerization), followed by one cycle of 4 min at 72°C, with final stabilization at 10°C. The amplified fragments were separated on. 1.5% agarose by gel ..... deforestation in the Cerrado biome (MMA 2014) and the extraction of B. capitata continues, and this species may soon cease to exist outside of ...

  4. Expression, purification and characterization of the interferon ...

    Indian Academy of Sciences (India)

    2012-01-19

    Jan 19, 2012 ... utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent ...

  5. Identification of differentially expressed genes in seeds of two - AJOL

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... cDNA subtraction Kit (Clontech) according to the manufacturer's protocols. To detect .... removal of vector, poor quality and polyA sequences). Sequence analysis ... suggests different activity of S-ACP-DES in the two lines at the different ... the products were resolved on 1.5% agarose gels. These are major ...

  6. Use of polymerase chain reaction for detection of Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Østergaard, Lars; Birkelund, Svend; Christiansen, Gunna

    1990-01-01

    A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved...

  7. Correlating Whole Brain Neural Activity with Behavior in Head-Fixed Larval Zebrafish.

    Science.gov (United States)

    Orger, Michael B; Portugues, Ruben

    2016-01-01

    We present a protocol to combine behavioral recording and imaging using 2-photon laser-scanning microscopy in head-fixed larval zebrafish that express a genetically encoded calcium indicator. The steps involve restraining the larva in agarose, setting up optics that allow projection of a visual stimulus and infrared illumination to monitor behavior, and analysis of the neuronal and behavioral data.

  8. Differentiation studies of predominant lactic acid bacteria isolated ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... Lactobacillus plantarum subsp. plantarum NBRC 15891 as a reference strain. But many .... with conserved primers close to the 3_ and 5_ ends of the gene. ... specific. Agarose gel electrophoresis was done to examine the ..... Lactobacillus delbrueckii subsp. delbrueckii LMG 6412T (AM087689). AA2. AA5.

  9. Genetic diversity in African nutmeg (Monodora myristica) accessions ...

    African Journals Online (AJOL)

    sunny

    2014-10-15

    Oct 15, 2014 ... The purity of DNA was determined by calculating the ratio of absorbance at 260 nm to that of 280 nm. DNA concentration and purity was also determined by running the samples on 0.8% agarose gel. Polymerase chain reactions (PCR) were carried out in a Peltier thermal cycler (BioRAD DNA engine) using ...

  10. Genetic diversity of pheasants from natural habitat and farm ...

    African Journals Online (AJOL)

    Roman

    2013-05-01

    May 1, 2013 ... ducted in a MJ Research PTC-225 Peltier Thermal Cycler. After the amplification, the product was separated with the electrophoretic method on 1.8% agarose gel with an admixture of ethidium bromide at 80 V for 210 min. Tagging with ethidium bromide as an intercalating agent consists in its excitation with ...

  11. (ROP2) gene of Toxoplasma gondii in eukaryotic cell

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... responsible for animal and human toxoplasmosis. In ... body and has also epitope for B-cell and produces IgA,. IgM and IgG (Saavedra et al., ... DNA extraction products were detected in 0.8% agarose gel and photographed.

  12. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    Science.gov (United States)

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  13. Fingerprinting of Fagaceae individuals using intermicrosatellite ...

    Indian Academy of Sciences (India)

    (sweet chestnut). 10. HVR20157 ... products were visualized after electrophoresis on 1.8% w/v agarose gels .... did not overestimate the heterozygosity, proving the ISSRs ability to ..... genus. In this study and in a previous work (Coutinho et al.

  14. A novel missense mutation of ADAR1 gene in a Chinese family ...

    Indian Academy of Sciences (India)

    This study was mainlyto explore the pathogenic mutation of ADAR1 gene and provide genetics counselling and prenatal diagnostic testing for childbearing individuals.Mutational analysis of ADAR1 gene was performed by polymerase chain reaction (PCR) and electrophoretic separation of PCR products by 1.5% agarose ...

  15. Detection of viable toxigenic Vibrio cholerae and virulent Shigella ...

    African Journals Online (AJOL)

    . cholerae and the invasion plasmid antigen gene (ipaH) of virulent Shigella spp., was performed and the PCR products were visualised by agarose gel electrophoresis. The assay allowed the detection of as few as 1 cfu/100 ml of V. cholerae ...

  16. modifying and adapting a plant-based dna extraction protocol for ...

    African Journals Online (AJOL)

    transferred into new eppendorf tubes and 200µl of cold isopropanol was added, inverted 5-6 ... The pellets were washed using. 500µl cold ethanol and centrifuged at10,000 r/min for 5- ... 2: Agarose gel electrophoresis showing the 850bp PCR product of the Angiotensin II type I receptor gene. (Lane. M is the 100bp DNA ...

  17. Identification of four genes involved in suppression of the pre-mRNA ...

    Indian Academy of Sciences (India)

    Unknown

    TTTTTTTTTTTTTTTTT3' and 5'CCAAGGCGATA-. TCGATATTTG3'. The PCR products were resolved by agarose gel electrophoresis and subjected to Southern blotting and hybridization (Sambrook et al. 1989) with. PCR-amplified rhp6 cDNA which was radiolabelled by the random primer method (Feinberg and Vogelstein.

  18. An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments

    DEFF Research Database (Denmark)

    Yu, Ran; Graf, Joerg; Smets, Barth F.

    2008-01-01

    Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using β...

  19. endotoxin genes of Bacillus thuringiensis by pulse field gel

    African Journals Online (AJOL)

    Owner

    Two of the isolates had only 1 band whereas the others had more .... g tryptone; 2 g tryptose; 1.5 g yeast extract; 0.05 M sodium .... of the ultra pure Seakem® Gold agarose gel which corresponded ... any where near the observed frequency.

  20. Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

    NARCIS (Netherlands)

    Griffini, P.; Vigorelli, E.; Bertone, V.; Freitas, I.; van Noorden, C. J.

    1994-01-01

    The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate

  1. RT-PCR and real-time PCR analysis of E2A-PBX1, TEL-AML1 ...

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... group of 1–25 years (50 pediatric and 14 young adults). Diagnosis of ... 3% agarose gel to visualize the amplified products of fusion gene transcripts. .... translocation in 13.4% (177/1321, range 9–23%) of child- hood ALL in Far ... to apoptosis, growth factor interactions and alters cell–cell and cell–matrix ...

  2. 50 Detecting adenosine triphosphatase 6 point mutations that may ...

    African Journals Online (AJOL)

    mutations at codons for the key residues Lys 260, Leu263, Gln266, Ser769 .... agarose gel and visualized under UV transillumination after treatment with ..... Li, W., Mo, W., Shen, D., Sun, L., Wang, J., Lu, S., Gitschier, J.M. & Zhou, B. (2005) Yeast ... Nagamune, K., Beatty, W.L., & Sibley, D. (2007) Artemisinin induces Calcium ...

  3. A novel inert crystal delivery medium for serial femtosecond crystallography

    Directory of Open Access Journals (Sweden)

    Chelsie E. Conrad

    2015-07-01

    Full Text Available Serial femtosecond crystallography (SFX has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

  4. relationship of status of polymorphic rapd bands with genotypic

    African Journals Online (AJOL)

    jen

    Department of Plant Breeding and Genetics, College of Agriculture, Orissa University of Agriculture and. Technology .... Amplified Polymorphic DNA) have been ... DNA quantification was done by visualising under UV light after electrophoresis on 0.8% (w/v) agarose gel. The. DNA was again diluted in TE buffer to 5 μg μl-1.

  5. Controlled transmission of African cassava mosaic virus (ACMV) by ...

    African Journals Online (AJOL)

    Sarkodie

    2013-07-10

    Jul 10, 2013 ... Box LG 80 Legon, Accra, Ghana. Accepted 1 July, 2013. Jatropha curcas, a plant with great biodiesel potential is also used to reduce the population of .... loading dye and electrophoresed on 1% agarose gel at 90 V for 1 h.

  6. HLA-DPB1 typing with polymerase chain reaction and restriction fragment length polymorphism technique in Danes

    DEFF Research Database (Denmark)

    Hviid, T V; Madsen, H O; Morling, N

    1992-01-01

    endonucleases: RsaI, FokI, ApaI, SacI, BstUI, EcoNI, and DdeI, and the DNA fragments were separated by electrophoresis in agarose gels. Altogether, 71 individuals were investigated and 16 different HLA-DPB1 types were observed in 26 different heterozygotic combinations, as well as five possible homozygotes...

  7. Gel Electrophoresis--The Easy Way for Students

    Science.gov (United States)

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

  8. Augmenting convection-enhanced delivery through simultaneous co-delivery of fluids and laser energy with a fiberoptic microneedle device

    Science.gov (United States)

    Hood, R. Lyle; Ecker, Tobias; Andriani, Rudy; Robertson, John; Rossmeisl, John; Rylander, Christopher G.

    2013-03-01

    This paper describes a new infusion catheter, based on our fiberoptic microneedle device (FMD), designed with the objective of photothermally augmenting the volumetric dispersal of infused therapeutics. We hypothesize that concurrent delivery of laser energy, causing mild localized photothermal heating (4-5 °C), will increase the spatial dispersal of infused chemotherapy over a long infusion period. Agarose brain phantoms, which mimic the brain's mechanical and fluid conduction properties, were constructed from 0.6 wt% Agarose in aqueous solution. FMDs were fabricated by adhering a multimode fiberoptic to a silica capillary tube, such that their flat-polished tips co-terminated. Continuous wave 1064 nm light was delivered simultaneously with FD&C Blue #2 (5%) dye into phantoms. Preliminary experiments, where co-delivery was tested against fluid delivery alone (through symmetrical infusions into in vivo rodent models), were also conducted. In the Agarose phantoms, volumetric dispersal was demonstrated to increase by more than 3-fold over a four-hour infusion time frame for co-delivery relative to infusion-only controls. Both forward and backward (reflux) infusions were also observed to increase slightly. Increased volumetric dispersal was demonstrated with co-delivery in an in vivo rodent model. Photothermal augmentation of infusion was demonstrated to influence the directionality and increase the volume of dye dispersal in Agarose brain phantoms. With further development, FMDs may enable a greater distribution of chemotherapeutic agents during CED therapy of brain tumors.

  9. Cryopreservation of organotypic multicellular spheroids from human gliomas

    NARCIS (Netherlands)

    Kaaijk, P.; van den Berg, F.; van Amstel, P.; Troost, D.

    1996-01-01

    Fresh human glioma tissue can be cultured on agarose to form organotypic multicellular spheroids (OMS). The major advantage of OMS is the preservation of the cellular heterogeneity and the tumour architecture, which is lost in conventional monolayer cultures. The present study was undertaken to

  10. Download this PDF file

    African Journals Online (AJOL)

    made visible by ethidium bromide. The results from gel electrophoresis were visualized on a UV transillumination. (254 nm). Agarose gel preparation as well as electrophoresis were carried out using buffer solution (pH 8.0), contain- ing 45 mmol/L Tris-base/boric acid and. 1 mmol/L EDTA adjusted with hydro- chloric acid.

  11. Genetic diversity analysis of some exotic, Indian and mutant ...

    African Journals Online (AJOL)

    USER

    2010-06-28

    Jun 28, 2010 ... 25 µl reaction mixture (reagents of a single PCR reaction given in. Table 3). Finally, the PCR tubes were subjected to the thermal profile given above. The reaction was carried out in a gradient. Mastercycler for amplification program. A 1.5% agarose gel in 1 X TAE buffer (Tris-base, glacial acetic acid, EDTA) ...

  12. Malignant transformation of guinea pig cells after exposure to ultraviolet-irradiated guinea pig cytomegalovirus

    International Nuclear Information System (INIS)

    Isom, H.C.; Mummaw, J.; Kreider, J.W.

    1983-01-01

    Guinea pig cells were malignantly transformed in vitro by ultraviolet (uv)-irradiated guinea pig cytomegalovirus (GPCMV). When guinea pig hepatocyte monolayers were infected with uv-irradiated GPCMV, three continuous epithelioid cell lines which grew in soft agarose were established. Two independently derived GPCMV-transformed liver cells and a cell line derived from a soft agarose clone of one of these lines induced invasive tumors when inoculated subcutaneously or intraperitoneally into nude mice. The tumors were sarcomas possibly derived from hepatic stroma or sinusoid. Transformed cell lines were also established after infection of guinea pig hepatocyte monolayers with human cytomegalovirus (HCMV) or simian virus 40 (SV40). These cell lines also formed colonies in soft agarose and induced sarcomas in nude mice. It is concluded that (i) GPCMV can malignantly transform guinea pig cells; (ii) cloning of GPCMV-transformed cells in soft agarose produced cells that induced tumors with a shorter latency period but with no alteration in growth rate or final tumor size; and (iii) the tumors produced by GPCMV-and HCMV-transformed guinea pig cells were more similar to each other in growth rate than to those induced by SV40-transformed guinea pig cells

  13. Modified Antifreeze Liquids for Use on Surfaces

    Science.gov (United States)

    Lynn, R. O.

    1983-01-01

    Report presents results of evaluation of two antifreeze liquids, dimethyl sulfoxide and ethylene glycol and five viscosity modifiers: gelatin, gum tragacanth, starch, agarose powder and citrus pectin. Purpose of evaluation to find best way of dealing with frost formation on Space Shuttle.

  14. Development of a competitive PCR assay for the quantification of ...

    African Journals Online (AJOL)

    ONOS

    2010-01-25

    Jan 25, 2010 ... quantification of total Escherichia coli DNA in water. Omar Kousar Banu, Barnard .... Thereafter the product was ligated into the pGEM®T-easy cloning ... agarose gel using the high pure PCR product purification kit. (Roche® ...

  15. Pulsed-field gel electrophoresis of bacterial chromosomes.

    Science.gov (United States)

    Mawer, Julia S P; Leach, David R F

    2013-01-01

    The separation of fragments of DNA by agarose gel electrophoresis is integral to laboratory life. Nevertheless, standard agarose gel electrophoresis cannot resolve fragments bigger than 50 kb. Pulsed-field gel electrophoresis is a technique that has been developed to overcome the limitations of standard agarose gel electrophoresis. Entire linear eukaryotic chromosomes, or large fragments of a chromosome that have been generated by the action of rare-cutting restriction endonucleases, can be separated using this technique. As a result, pulsed-field gel electrophoresis has many applications, from karyotype analysis of microbial genomes, to the analysis of chromosomal strand breaks and their repair intermediates, to the study of DNA replication and the identification of origins of replication. This chapter presents a detailed protocol for the preparation of Escherichia coli chromosomal DNA that has been embedded in agarose plugs, digested with the rare-cutting endonuclease NotI, and separated by contour-clamped homogeneous field electrophoresis. The principles in this protocol can be applied to the separation of all fragments of DNA whose size range is between 40 kb and 1 Mb.

  16. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as their HS DNA cleavage properties by means of agarose gel electrophoresis. The experimental results show that all the complexes can bind to DNA via partial intercalative mode.

  17. Phylogenetic analysis of the genus Anabaena based on PCR ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... LTRR2. 5'- CTA TCA GGG ATT GAA AG-3'. Rasmussen and. Svenning, 1998 on a 1% agarose gel containing 0.5 X TBE (Tris Borate-EDTA) and. 0.5 µg/ml ethidium bromide. Data analysis. Fingerprints generated from different Anabaena species were compared and all bands were scored. The presence or ...

  18. Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees.

    Science.gov (United States)

    Larson, Nicholas R; Anderson, Troy D

    2017-06-12

    The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

  19. Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein

    DEFF Research Database (Denmark)

    Gram, Trine; Ahrens, Peter

    1998-01-01

    species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity...

  20. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Synthesis of agarose-metal/semiconductor nanoparticles having superior bacteriocidal activity and their simple conversion to metal-carbon composites ... Chemistry and Physics of Materials Unit and DST Unit on Nanoscience, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur PO, Bangalore 560 064 ...

  1. germplasm using ISSR markers and their relationships

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... (MAS) is the current trend in 'Modern Agriculture'. These. DNA markers allow the construction of ... are inherited in Mendelian fashion and are scored as dominant markers (Ratnaparkhe et al., 1998) ... ISSR amplified PCR products were resolved on 2% agarose gel in. 1X TBE buffer (89 mM Tris-Hcl, pH 8.3, ...

  2. One-step purification of E. coli elongation factor Tu

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.; Degn, B

    1993-01-01

    The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one...

  3. Understanding Electrophoresis through the Investigation of Size, Shape, and Charge of pH Indicators

    Science.gov (United States)

    Brenner, Ryan K.; Hess, Kenneth R.; Morford, Jennifer L.

    2015-01-01

    A laboratory experiment was designed for upper-level students in a Chemical Analysis course to illustrate the theoretical and practical applications of 0.8% agarose gel electrophoresis and to reinforce an understanding of weak acids/bases using easy-to-visualize pH indicators. The careful choice of indicators included acid and base types with…

  4. Induction of protective immune responses in mice by double DNA ...

    African Journals Online (AJOL)

    Keywords: Multiple DNA vaccine, Omp31 gene, Brucella melitensis, Eae gene, Escherichia ... Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African .... a 1 % agarose gel in 1× TBE buffer, followed by ... manufacturer's protocol, the recombinant ..... Moreno S, Timon M. DNA vaccination: an immunological.

  5. Fast and non-invasive PCR sexing of primates: apes, Old World monkeys, New World monkeys and Strepsirrhines

    DEFF Research Database (Denmark)

    Villesen, Palle; Fredsted, Tina

    2006-01-01

    is described using a triple primer PCR reaction and agarose gel electrophoresis of the sex-chromosomal isoforms of the ubiquitously transcribed tetratricopeptide repeat protein gene (UTX/UTY). By comparing genomic data from several mammals we identified the UTX/UTY locus as the best candidate for a universal...

  6. Diffusion measurement in ferrous infused gel dosimeters

    International Nuclear Information System (INIS)

    Zahmatkesh, M. H.; Healy, B. J.

    2003-01-01

    Background: The compositions of Ferrous sulphate, Agarose and Xylenol orange dye and Ferrous sulphate, Gelatin and Xylenol orange dye in solution of distilled water and sulphuric acid are two tissue-equivalent gel dosimeters. Ionizing radiation causes oxidation of Fe 2+ ion to Fe 3+ ions which diffuse through the gel matrix and blur the image of absorbed dose over a period of hours after irradiation. Materials and methods: 25 m M sulphuric acid, 0.4 mm ferrous ammonium sulphate, 0.2 mm xylenol orange dye and 1% by weight agarose in distilled water named Agarose and Xylenol orange dye and 0.1 mm ferrous ammonium sulphate, 0.1 mm xylenol orange dye, 50 mm sulphuric acid and 5% by weight gelatin in distilled water named Gelatin and Xylenol orange dye are used as two gel dosimeters. All chemicals were supplied by Sigma Ald ridge Company, Germany. The gels were poured in Perspex casts and were irradiated to a beam of X ray from linear accelerators or X ray machine. Results: In this study diffusion coefficients of Agarose and Xylenol orange dye and Gelatin and Xylenol orange dye dosimeters have been measured through a computer program for different temperature. The ferric ion diffusion coefficient (D) for the Agarose and Xylenol orange dye and Gelatin and Xylenol orange dye dosimeters were measured as (1.19.±0.03) x 10 -2 cm 2 .hr -1 and (0.83±0.03) x 10 -2 cm 2 .hr -1 respectively at room temperature. Conclusion: For both dosimeters the diffusion coefficients decreased with gel storage temperatures down to 6 d ig C . Gelatin and Xylenol orange dye dosimeters have advantage of lower diffusion coefficient for a specified temperature

  7. MR thermometry for laser-induced thermotherapy at 1.5 tesla; MR-Thermometrie bei 1,5 Tesla zur thermischen Ablation mittels laserinduzierter Thermotherapie

    Energy Technology Data Exchange (ETDEWEB)

    Meister, D.; Huebner, F.; Mack, M.; Vogl, T.J. [Frankfurt Univ. (Germany). Inst. fuer Diagnostische und Interventionelle Radiologie

    2007-05-15

    Purpose: Evaluation of thermometry with fast MR sequences for laser-induced interstitial laser therapy (LITT) and verification of the thermometric results with a fiber-optic thermometer. Method and Materials: In vitro experiments were conducted using an agarose gel mixture and pig liver lobes. MR-guided LITT was performed using a laser power between 3 and 15?watts. Thermometry was performed using longitudinal relaxation time T1 and proton resonance frequency shift (PRF) methods under acquisition of amplitude and phase shift images. PRF was measured with a fast spoiled GRE sequence. Four different sequences were used for T1 thermometry: gradient echo (GE), TrueFISP (TRUFI), Saturation Recovery Turbo-FLASH (SRTF) and Inversion Recovery Turbo-FLASH (IRTF) sequences. The temperature was controlled using a fiber-optic Luxtron device and correlated with the MR temperature. The range of applied and monitored temperatures exceeded 80 degrees Celsius. Results: The temperature dependence showed a good linear relationship up to 60 degrees Celsius. Calibration experiments for the T1 method delivered coefficients of determination from 0.977 to 0.997 for agarose and from 0.958 to 0.995 for the pig liver samples. The IRTF sequence had the highest temperature sensitivity (agarose 0.99, liver 1.19). During LITT the TRUE-FISP sequence exhibited a strong nonlinear relationship. R{sup 2} of this sequence was 0.809 in the agarose experiments. The average temperature errors when heated up to 80 degrees Celsius were 3.86 - 11.38 degrees Celsius for Agarose gel and 5.7 - 12.16 degrees Celsius for the liver tissue. SRTF and IRTF sequences exhibited the most linear relationship with temperature but were more dependent on tissue differences. (orig.)

  8. Blot hybridization analysis of TCR genes of T cells for five people exposed in a radiation accident

    International Nuclear Information System (INIS)

    Min Rui; Liu Benti; Cheng Tianmin; Yang Rujun; Meng Xiangshun; Xiao Jinsong

    1996-01-01

    Human lymphocyte total DNA was prepared in agarose plug by mixing cells with low melting agarose, and two restriction endonucleases were used for digestion of the total DNA with human α and β TCR cDNA probes. The total digested DNA from five people who were whole body exposed to 2.0-2.5 Gy ionizing radiation in an accident 4.5 years ago was hybridized by Southern blot method. The results showed that no obvious difference in hybridization bands was found between controls and the five victims when hybridizations were fulfilled in the total DNA which was digested by Hind III restriction endonuclease with both α and β probes. However, when the total DNA was digested with restriction endonuclease EcoR I and was hybridized with TCR α probe, four of the five exposed people showed a different hybridizing band pattern compared with the controls. The results are also discussed

  9. Comprehensive Study of Microgel Electrode for On-Chip Electrophoretic Cell Sorting

    Science.gov (United States)

    Hattori, Akihiro; Yasuda, Kenji

    2010-06-01

    We have developed an on-chip cell sorting system and microgel electrode for applying electrostatic force in microfluidic pathways in the chip. The advantages of agarose electrodes are 1) current-driven electrostatic force generation, 2) stability against pH change and chemicals, and 3) no bubble formation caused by electrolysis. We examined the carrier ion type and concentration dependence of microgel electrode impedance, and found that CoCl2 has less than 1/10 of the impedance from NaCl, and the reduction of the impedance of NaCl gel electrode was plateaued at 0.5 M. The structure control of the microgel electrode exploiting the surface tension of sol-state agarose was also introduced. The addition of 1% (w/v) trehalose into the microgel electrode allowed the frozen storage of the microgel electrode chip. The experimental results demonstrate the potential of our system and microgel electrode for practical applications in microfluidic chips.

  10. Human complement component C3

    DEFF Research Database (Denmark)

    Behrendt, N

    1985-01-01

    The two common genetic variants of human C3, C3 S and C3 F, were purified and characterized by SDS-PAGE, agarose gel electrophoresis, isoelectric focusing and amino acid analysis. The difference in electrophoretic mobility between the two variants was conserved after purification, and by isoelect......The two common genetic variants of human C3, C3 S and C3 F, were purified and characterized by SDS-PAGE, agarose gel electrophoresis, isoelectric focusing and amino acid analysis. The difference in electrophoretic mobility between the two variants was conserved after purification......, and by isoelectric focusing of the hemolytically active proteins, pI values of 5.86 and 5.81 were determined for C3 S and C3 F, respectively. Any difference in amino acid composition was too small to be detected by amino acid analysis, and the two proteins had the same molecular weight as determined by SDS-PAGE....

  11. In vitro evaluation of biodegradable microspheres with surface-bound ligands.

    Science.gov (United States)

    Keegan, Mark E; Royce, Sara M; Fahmy, Tarek; Saltzman, W Mark

    2006-02-21

    Protein ligands were conjugated to the surface of biodegradable microspheres. These microsphere-ligand conjugates were then used in two in vitro model systems to evaluate the effect of conjugated ligands on microsphere behavior. Microsphere retention in agarose columns was increased by ligands on the microsphere surface specific for receptors on the agarose matrix. In another experiment, conjugating the lectin Ulex europaeus agglutinin 1 to the microsphere surface increased microsphere adhesion to Caco-2 monolayers compared to control microspheres. This increase in microsphere adhesion was negated by co-administration of l-fucose, indicating that the increase in adhesion is due to specific interaction of the ligand with carbohydrate receptors on the cell surface. These results demonstrate that the ligands conjugated to the microspheres maintain their receptor binding activity and are present on the microsphere surface at a density sufficient to target the microspheres to both monolayers and three-dimensional matrices bearing complementary receptors.

  12. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  13. Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Wlodek, D.; Banath, J.; Olive, P.L.

    1991-01-01

    Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5Gy of radiation. Low voltage (ca.0.6V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage. (author)

  14. Role of electrostatic interactions on the transport of druglike molecules in hydrogel-based articular cartilage mimics

    DEFF Research Database (Denmark)

    Ye, Fengbin; Baldursdottir, Stefania G.; Hvidt, Søren

    2016-01-01

    In the field of drug delivery to the articular cartilage, it is advantageous to apply artificial tissue models as surrogates of cartilage for investigating drug transport and release properties. In this study, artificial cartilage models consisting of 0.5% (w/v) agarose gel containing 0.5% (w...... to the pure agarose gel. The decrease in apparent diffusivity of the cationic compounds was not caused by a change in the gel structure since a similar reduction in apparent diffusivity was not observed for the net negatively charged protein α-lactalbumin. The apparent diffusivity of the cationic compounds...... the electrostatic nature of their interactions. The results obtained from the UV imaging diffusion studies are important for understanding the effect of drug physicochemical properties on the transport in articular cartilage. The extracted information may be useful in the development of hydrogels for in vitro...

  15. Lectin affinity electrophoresis.

    Science.gov (United States)

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  16. Numerical modelling and experimental study of liquid evaporation during gel formation

    Science.gov (United States)

    Pokusaev, B. G.; Khramtsov, D. P.

    2017-11-01

    Gels are promising materials in biotechnology and medicine as a medium for storing cells for bioprinting applications. Gel is a two-phase system consisting of solid medium and liquid phase. Understanding of a gel structure evolution and gel aging during liquid evaporation is a crucial step in developing new additive bioprinting technologies. A numerical and experimental study of liquid evaporation was performed. In experimental study an evaporation process of an agarose gel layer located on Petri dish was observed and mass difference was detected using electronic scales. Numerical model was based on a smoothed particle hydrodynamics method. Gel in a model was represented as a solid-liquid system and liquid evaporation was modelled due to capillary forces and heat transfer. Comparison of experimental data and numerical results demonstrated that model can adequately represent evaporation process in agarose gel.

  17. QNS measurements on water in biological and model systems

    International Nuclear Information System (INIS)

    Trantham, E.C.; Rorschach, H.E.; Clegg, J.C.; Hazlewood, C.F.; Nicklow, R.M.

    1981-01-01

    Results are presented on the quasi-elastic spectra of 0.95 THz neutrons scattered from pure water, a 20% agarose gel and cysts of the brine shrimp (Artemia) of hydration 1.2 gms H 2 O per gm of dry solids. The lines are interpreted with a two-component model in which the hydration water scatters elastically and the free water is described by a jump-diffusion correlation function. The results for the line widths GAMMA(Q 2 ) are in good agreement with previous measurements for the water sample but show deviations from pure water at large Q for agarose and the Artemia cysts that suggest an increased value of the residence time in the jump-diffusion model

  18. Optimization of the southern electrophoretic transfer method

    International Nuclear Information System (INIS)

    Allison, M.A.; Fujimura, R.K.

    1987-01-01

    The technique of separating DNA fragments using agarose gel electrophoresis is essential in the analysis of nucleic acids. Further, after the method of transferring specific DNA fragments from those agarose gels to cellulose nitrate membranes was developed in 1975, a method was developed to transfer DNA, RNA, protein and ribonucleoprotein particles from various gels onto diazobenzyloxymethyl (DBM) paper using electrophoresis as well. This paper describes the optimum conditions for quantitative electrophoretic transfer of DNA onto nylon membranes. This method exemplifies the ability to hybridize the membrane more than once with specific RNA probes by providing sufficient retention of the DNA. Furthermore, the intrinsic properties of the nylon membrane allow for an increase in the efficiency and resolution of transfer while using somewhat harsh alkaline conditions. The use of alkaline conditions is of critical importance since we can now denature the DNA during transfer and thus only a short pre-treatment in acid is required for depurination. 9 refs., 7 figs

  19. Improved DNA electrophoresis in conditions favoring polyborates and lewis acid complexation.

    Directory of Open Access Journals (Sweden)

    Hari Singhal

    2010-06-01

    Full Text Available Spatial compression among the longer DNA fragments occurs during DNA electrophoresis in agarose and non-agarose gels when using certain ions in the conductive buffer, impairing the range of fragment sizes resolved well in a single gel. Substitutions using various polyhydroxyl anions supported the underlying phenomenon as the complexation of Lewis acids to DNA. We saw significant improvements using conditions (lithium borate 10 mM cations, pH 6.5 favoring the formation of borate polyanions and having lower conductance and Joule heating, delayed electrolyte exhaustion, faster electrophoretic run-speed, and sharper separation of DNA bands from 100 bp to 12 kb in a single run.

  20. [Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation].

    Science.gov (United States)

    Li, Jinlong; Zhang, Yidan; Zhou, Xin; Ji, Wenjie; Zhao, Jihong; Wei, Luqing; Li, Yuming

    2014-09-01

    To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis. Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen. HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees. This work established an efficient method for in vitro generation and degradation of human NETs.

  1. BHC80 is Critical in Suppression of Snail-LSD1 Interaction and Breast Cancer Metastasis

    Science.gov (United States)

    2014-04-01

    Form Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including...or H3K4me2 antibody overnight, followed by incubation with a 50% slurry of protein A–agarose/Salmon 22 sperm DNA (Upstate Biotechnology, Lake...manufacturer’s protocol (Applied Biosystems). MTT assay MTT assays were performed using standard protocol. Cell count and incubation time were optimized

  2. Photo-Acoustic Ultrasound Imaging to Distinguish Benign from Malignant Prostate Cancer

    Science.gov (United States)

    2016-09-01

    tissue phantoms and animal models of disease . 15. SUBJECT TERMS Photoacoustic, Ultrasound imaging, transurethral probe 16. SECURITY CLASSIFICATION...visible, ultrasound images are unable to discriminate between benign or malignant cancers. In photoacoustic imaging, laser energy is transmitted ...40 g/L concentration of sea plaque agarose into DI water heated to approximately 80°C. A 10 g/L concentration of silica powder was then added to

  3. Purification and properties of enolase of human erythrocytes

    NARCIS (Netherlands)

    Hoorn, R.K.J.; Flikweert, J.P.; Staal, Gerard E.J.

    1974-01-01

    1. 1. Human erythrocyte enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was purified I000-fold. 2. 2. The pH-optimum was at pH 6.5. The molecular weight, estimated by gel filtration, was found to be 95,000 ± 5,000. 3. 3. Electrophoresis on agar-agarose at pH 8.5 and 6.4 showed only one

  4. The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    Science.gov (United States)

    Gurd, J W; Bissoon, N

    1997-08-01

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

  5. Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

    OpenAIRE

    Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

    2007-01-01

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the fi rst-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic...

  6. Colletotrichum circinans and Colletotrichum coccodes can be ...

    African Journals Online (AJOL)

    Administrator

    2003-12-29

    Dec 29, 2003 ... coccodes (DSM 62126, 66376, 70882, 70879, DSM. 2492, 70880) and C. circinans (67846 and ... µl 2.0 mM dNTP mixtures, 0.5 µl DMSO (2% v/v), 0.5 µl each of the primers at 10pmol concentration, 0.25 µl ... analysed by electrophoresis in 1.5% (w/v) agarose gels and ethidium bromide staining. provided ...

  7. Genetic identification of new alien pest species Illinoia liriodendri and its parasitoid Areopraon silvestre in Croatia

    OpenAIRE

    Franjević, Milivoj; Glavaš, Milan; Hrašovec, Boris; Koletić, Nikola; Franjević, Damjan

    2016-01-01

    Background and Purpose: During June 2015 in Zagreb city area (Croatia) samples of tulip tree (Liriodendron tulipifera) leaves were collected with symptoms of attack by some unknown aphid. Material and methods: Aphids were collected form leaves of tulip trees on different locations in Zagreb during July 2015. Total genomic DNA was extracted from ethanol-preserved specimens. PCR analysis was carried out and PCR products were purified from 1% agarose gel for sequencing purposes. The obtaine...

  8. Polymerase chain reaction for detection of invasive Shigella flexneri in food.

    OpenAIRE

    Lampel, K A; Jagow, J A; Trucksess, M; Hill, W E

    1990-01-01

    The polymerase chain reaction (PCR) was used to amplify a 760-base-pair (bp) fragment with the 220-kbp invasive plasmids of enteroinvasive Escherichia coli, Shigella flexneri, Shigella dysenteriae, Shigella boydii, and Shigella sonnei as templates. This PCR product was easily detected by agarose gel electrophoresis. A 210-bp AccI-PstI fragment lying within the amplified region was used as a probe in Southern hybridization blots and showed that the PCR-generated product was derived from the in...

  9. Different sterilization methods for overcoming internal bacterial infection in sunflower seeds

    Directory of Open Access Journals (Sweden)

    Taški-Ajduković Ksenija J.

    2005-01-01

    Full Text Available During culture of protoplasts in agarose droplets, permanent problem was bacterial infection. It was assumed that the seeds are the origin of infection, so different sterilization methods were tested in order to overcome this problem. Germination, infection of seeds and hypocotyls and their growth were examined. Based on these parameters, the best result was obtained with the combined use of 5% commercial bleach and dry heating at 45°C.

  10. Physiological and Growth Characteristics of Shewanella Species

    Science.gov (United States)

    2016-05-01

    with B vitamins in the MMB. After the addition of riboflavin, DPV scans revealed a peak at -0.419 ± 0.005 V; n = 3. DPV scans performed on CF 31 h...0.008. The DM was supplemented with Wolfe’s mineral and vitamin solutions [20]. Peptone and yeast extract were omitted and replaced with high-purity...containing MB, cell elongation was observed when cultures entered stationary phase. Under DM conditions, agarose was in excess throughout the

  11. Integrated Microanalytical System for Simultaneous Voltammetric Measurements of Free Metal Ion Concentrations in Natural Waters

    OpenAIRE

    Noël, Stéphane; Tercier-Waeber, Mary-Lou; Lin, Lin; Buffle, Jacques; Guenat, Olivier; Koudelka-Hep, Milena

    2007-01-01

    A complexing gel integrated microelectrode (CGIME) for direct measurements of free metal ion concentrations in natural waters has been developed. It is prepared by the successive deposition of microlayers of a chelating resin, an antifouling agarose gel and Hg on a 100-interconnected Ir-based microelectrode array. The trace metals of interest are in a first step accumulated on the chelating resin in proportion to their free ion concentration in solution, then released in acidic solution and d...

  12. Simple practical approach for sample loading prior to DNA extraction using a silica monolith in a microfluidic device.

    Science.gov (United States)

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-12-07

    A novel DNA loading methodology is presented for performing DNA extraction on a microfluidic system. DNA in a chaotropic salt solution was manually loaded onto a silica monolith orthogonal to the subsequent flow of wash and elution solutions. DNA was successfully extracted from buccal swabs using electro-osmotic pumping (EOP) coupled with in situ reagents contained within a 1.5% agarose gel matrix. The extracted DNA was of sufficient quantity and purity for polymerase chain reaction (PCR) amplification.

  13. Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

    International Nuclear Information System (INIS)

    Hamilton, R.G.; Rendell, M.; Adkinson, N.F. Jr.

    1980-01-01

    A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively

  14. Characterization of marine bacteria highly resistant to mercury exhibiting multiple resistances to toxic chemicals

    Digital Repository Service at National Institute of Oceanography (India)

    De, J.; Ramaiah, N.

    , GP15 and GP16) and one Pseudomonas aeruginosa (CH07) which showed comparatively higher resistance to toxic heavy metals and xenobiotics and were used in more detailed experiments. Antibiotic sensitivity of all three isolates after plasmid curing... using Nucleospin Plasmid isolation kit (Macherey Nagel, Germany) and agarose gel electrophoresis. To further confirm the presence/absence of plasmid, two different plasmid curing assays were performed to note the loss, if any, of mercury resistance...

  15. Novel Protection and Decontamination Strategies

    Science.gov (United States)

    2016-06-01

    agents and toxins that can be applied to both civilian and military use. The work can be divided into investigations into two broad topic areas...work can be divided into investigations into two broad, basic science topic areas: development of novel rationally designed virus inactivation...again in PBS and agarose overlays, incubation, and immunological detection was conducted as described for the focus forming unit assay. Virus:cell

  16. Low cost, low tech SNP genotyping tools for resource-limited areas: Plague in Madagascar as a model.

    Science.gov (United States)

    Mitchell, Cedar L; Andrianaivoarimanana, Voahangy; Colman, Rebecca E; Busch, Joseph; Hornstra-O'Neill, Heidie; Keim, Paul S; Wagner, David M; Rajerison, Minoarisoa; Birdsell, Dawn N

    2017-12-01

    Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs. To demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar), we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM). We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort. The success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable financial cost for

  17. Biological Influence of Deuterium on Procariotic and Eukaryotic Cells

    OpenAIRE

    Oleg Mosin; Ignat Ignatov

    2014-01-01

    Biologic influence of deuterium (D) on cells of various taxonomic groups of prokaryotic and eukaryotic microorganisms realizing methylotrophic, chemoheterotrophic, photo-organotrophic, and photosynthetic ways of assimilation of carbon substrates are investigated at growth on media with heavy water (D2О). The method of step by step adaptation technique of cells to D2О was developed, consisting in plating of cells on 2 % agarose nutrient media containing increasing gradient of concentration of ...

  18. Pattern transition between periodic Liesegang pattern and crystal growth regime in reaction-diffusion systems

    Science.gov (United States)

    Lagzi, István; Ueyama, Daishin

    2009-01-01

    The pattern transition between periodic precipitation pattern formation (Liesegang phenomenon) and pure crystal growth regimes is investigated in silver nitrate and potassium dichromate system in mixed agarose-gelatin gel. Morphologically different patterns were found depending on the quality of the gel, and transition between these typical patterns can be controlled by the concentration of gelatin in mixed gel. Effect of temperature and hydrodynamic force on precipitation pattern structure was also investigated.

  19. Antibacterial Compounds from Red Seaweeds (Rhodophyta)

    OpenAIRE

    Noer Kasanah; Triyanto Triyanto; Drajad Sarwo Seto; Windi Amelia; Alim Isnansetyo

    2015-01-01

    Seaweeds produce great variety of metabolites benefit for human. Red seaweeds (Rhodophyta) are well known as producer of phycocolloids such agar, agarose, carragenan and great variety of secondary metabolites. This review discusses the red algal secondary metabolites with antibacterial activity. The chemical constituents of red algae are steroid, terpenoid, acetogenin and dominated by halogenated compounds mainly brominated compounds. Novel compounds with intriguing skeleton are also reported...

  20. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  1. Culturable bacterial phylogeny from a shallow water hydrothermal vent of Espalamaca (Faial, Azores) reveals a variety of novel taxa

    Digital Repository Service at National Institute of Oceanography (India)

    Rajasabapathy, R.; Mohandass, C.; Colaco, A.; Dastager, S.G.; Santos, R.S.; Meena, R.M.

    by 1% agarose gel elec- trophoresis with TAE buffer. The PCR products were gel- purified using a Gel Extraction Kit or purified with PCR cleanup kit (Sigma) according to the manufacturer’s instructions. The purified PCR products were sequenced... productivity at the deep sea hydrothermal vents (> 200 m) is not maintained by photosynthetic products, but by the chemosynthesis of organic matter by vent microbes, using energy from chemical oxidation to produce organic matter from CO2 and mineral...

  2. Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

    OpenAIRE

    Vescovo, M; Morelli, L; Bottazzi, V

    1982-01-01

    Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined.

  3. Targeting histone abnormality in triple negative breast cancer

    Science.gov (United States)

    2017-08-01

    mass spectrometry analysis. Briefly, we used the biotinylated primers to prepare longer double stranded biotinylated HDAC5 promoter probes...streptavidin-agarose bead suspension was added to a mixture of 400 µg of nuclear extract proteins and 4 µg of double- strand biotinylated oligonucleotides in...20 Wu Y, Wang Y, Yang XH, Kang T, Zhao Y, Wang C et al. The deubiquitinase USP28 stabilizes LSD1 and confers stem -cell-like traits to breast cancer

  4. Population Based Assessment of MHC Class I Antigens Down Regulation as Markers of Increased Risk for Development and Progression of Breast Cancer from Benign Breast Lesions

    Science.gov (United States)

    2007-01-01

    Orchid Diagnostics Europe, St Katelijne Waver, Belgium). Sequence based typing In cases were complete HLA-losses on tumor cells were detected by...Centricon YM-100 filters (Millipore, Brussels, Belgium) and visualized in agarose gel electrophoresis using ethidiumbromide to show the single 2 KB band ...reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood. 1998;92:574-588. 22. Gao L, Bellantuono

  5. GLO polymorphism in Iceland

    Energy Technology Data Exchange (ETDEWEB)

    Karlsson, S; Arnason, A; Jensson, O

    1980-01-01

    The phenotypes of red cell glyoxalase I (GLO) were determined in two Icelandic population samples using starch-gel electrophoresis and high-voltage agarose-gel electrophoresis. The gene frequencies of 178 unrelated individuals were 0.46 for GLO/sup 1/ and 0.54 for GLO/sup 2/. In a group of Icelandic insulin-dependent diabetics the gene frequencies were found to be very similar. The evaluation of 30 mother-child pairs is also shown.

  6. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    OpenAIRE

    Hubbard Alan E; Dorsey Grant; Gupta Vinay; Rosenthal Philip J; Greenhouse Bryan

    2010-01-01

    Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary elec...

  7. Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods.

    OpenAIRE

    Shawar, R M; el-Zaatari, F A; Nataraj, A; Clarridge, J E

    1993-01-01

    Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkalin...

  8. Bioactive glass 13-93 as a subchondral substrate for tissue-engineered osteochondral constructs: a pilot study.

    Science.gov (United States)

    Jayabalan, Prakash; Tan, Andrea R; Rahaman, Mohammed N; Bal, B Sonny; Hung, Clark T; Cook, James L

    2011-10-01

    Replacement of diseased areas of the joint with tissue-engineered osteochondral grafts has shown potential in the treatment of osteoarthritis. Bioactive glasses are candidates for the osseous analog of these grafts. (1) Does Bioactive Glass 13-93 (BG 13-93) as a subchondral substrate improve collagen and glycosaminoglycan production in a tissue-engineered cartilage layer? (2) Does BG 13-93 as a culture medium supplement increase the collagen and glycosaminoglycan production and improve the mechanical properties in a tissue-engineered cartilage layer? In Study 1, bioactive glass samples (n = 4) were attached to a chondrocyte-seeded agarose layer to form an osteochondral construct, cultured for 6 weeks, and compared to controls. In Study 2, bioactive glass samples (n = 5) were cocultured with cell-seeded agarose for 6 weeks. The cell-seeded agarose layer was exposed to BG 13-93 either continuously or for the first or last 2 weeks in culture or had no exposure. Osteochondral constructs with a BG 13-93 base had improved glycosaminoglycan deposition but less collagen II content. Agarose scaffolds that had a temporal exposure to BG 13-93 within the culture medium had improved mechanical and biochemical properties compared to continuous or no exposure. When used as a subchondral substrate, BG 13-93 did not improve biochemical properties compared to controls. However, as a culture medium supplement, BG 13-93 improved the biochemical and mechanical properties of a tissue-engineered cartilage layer. BG 13-93 may not be suitable in osteochondral constructs but could have potential as a medium supplement for neocartilage formation.

  9. Synthesis of a nanocomposite biomaterial for implant tissue engineering

    OpenAIRE

    Santos Montes, Angélica

    2015-01-01

    In order to improve health and quality of life, the challenge to develop new biomaterials has become extremely relevant. In this project, our main objective is to obtain a nanocomposite biopolymer that serves as a temporal synthetic extracellular matrix for cell growth and tissue regeneration. This matrix consists of a hydrogel lm of chitosan or agarose doped with di erent ceramic nanoparticles: titanium dioxide (TiO2) and aluminum oxide (Al2O3). Once developed, this composite will be tested...

  10. The significance of gtf genes in caries expression: a rapid identification of Streptococcus mutans from dental plaque of child patients.

    Science.gov (United States)

    Mishra, Apurva; Pandey, Ramesh K; Manickam, Natesan

    2015-01-01

    Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.

  11. Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

    International Nuclear Information System (INIS)

    Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

    1988-01-01

    Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor α subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[γ- 32 P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor β subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins

  12. Interactions of trace metals with hydrogels and filter membranes used in DET and DGT techniques.

    Science.gov (United States)

    Garmo, Oyvind A; Davison, William; Zhang, Hao

    2008-08-01

    Equilibrium partitioning of trace metals between bulk solution and hydrogels/filter was studied. Under some conditions, trace metal concentrations were higher in the hydrogels or filter membranes compared to bulk solution (enrichment). In synthetic soft water, enrichment of cationic trace metals in polyacrylamide hydrogels decreased with increasing trace metal concentration. Enrichment was little affected by Ca and Mg in the concentration range typically encountered in natural freshwaters, indicating high affinity but low capacity binding of trace metals to solid structure in polyacrylamide gels. The apparent binding strength decreased in the sequence: Cu > Pb > Ni approximately to Cd approximately to Co and a low concentration of cationic Cu eliminated enrichment of weakly binding trace metal cations. The polyacrylamide gels also had an affinity for fulvic acid and/or its trace metal complexes. Enrichment of cationic Cd in agarose gel and hydrophilic polyethersulfone filter was independent of concentration (10 nM to 5 microM) but decreased with increasing Ca/ Mg concentration and ionic strength, suggesting that it is mainly due to electrostatic interactions. However, Cu and Pb were enriched even after equilibration in seawater, indicating that these metals additionally bind to sites within the agarose gel and filter. Compared to the polyacrylamide gels, agarose gel had a lower affinity for metal-fulvic complexes. Potential biases in measurements made with the diffusive equilibration in thin-films (DET) technique, identified by this work, are discussed.

  13. Effects of estrogen on very low-density lipoprotein triglyceride metabolism in fed and fasted chicks

    International Nuclear Information System (INIS)

    Park, J.R.

    1988-01-01

    A single injection of estrogen into growing chicks resulted in a marked elevation in plasma triglyceride (TG) followed by phospholipid (PL) and cholesterol (CH) in both fed and fasted chicks. Estrogen caused a development of massive fatty liver in fed chicks. Hepatic malic enzyme and glucose-6-phosphate dehydrogenase activities also increased significantly in fed chicks and, to a small extent, in fasted chicks. Very low density lipoproteins (VLDL) were barely detectable in the fasted control plasma. However, the VLDL concentration increased markedly upon estrogen injection, becoming the most prevalent lipoprotein in the plasma. The administration of estrogen resulted in an increase in oleic acid and a decrease in linoleic acid content except in the cholesteryl ester of VLDL and LDL. VLDL of estrogenized birds had β-mobility on agarose gel electrophoresis, and they eluted in two peaks on agarose gel filtration chromatography. Both peaks on gel filtration exhibited the same β-mobility on agarose gel electrophoresis. Nevertheless, the apoprotein composition of these two peaks were substantially different from each other; apo B was not present in the first peak VLDL. VLDL-TG kinetic studies conducted in vivo, using 14 C-TG-VLDL prepared endogenously from control and estrogenized chicks revealed that VLDL-TG produced from the former had a higher fractional catabolic rate (FCR) than VLDL-TG from the latter

  14. Quantification of specific bindings of biomolecules by magnetorelaxometry

    Directory of Open Access Journals (Sweden)

    Steinhoff Uwe

    2008-03-01

    Full Text Available Abstract The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP, to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX. Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality. The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads.

  15. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  16. The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

    International Nuclear Information System (INIS)

    Balart, Josep; Pueyo, Gemma; Llobet, Lara I de; Baro, Marta; Sole, Xavi; Marin, Susanna; Casanovas, Oriol; Mesia, Ricard; Capella, Gabriel

    2011-01-01

    Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay

  17. The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

    Science.gov (United States)

    Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

    2017-05-01

    Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

    Science.gov (United States)

    Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

    2016-03-01

    Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Patterning Multi-Nanostructured Poly(l-lactic acid) Fibrous Matrices to Manipulate Biomolecule Distribution and Functions.

    Science.gov (United States)

    Xiao, Wenwu; Li, Qingtao; He, Huimin; Li, Wenxiu; Cao, Xiaodong; Dong, Hua

    2018-03-14

    Precise manipulation of biomolecule distribution and functions via biomolecule-matrix interaction is very important and challenging for tissue engineering and regenerative medicine. As a well-known biomimetic matrix, electrospun fibers often lack the unique spatial complexity compared to their natural counterparts in vivo and thus cannot deliver fully the regulatory cues to biomolecules. In this paper, we report a facile and reliable method to fabricate micro- and nanostructured poly(l-lactic acid) (PLLA) fibrous matrices with spatial complexity by a combination of advanced electrospinning and agarose hydrogel stamp-based micropatterning. Specifically, advanced electrospinning is used to construct multi-nanostructures of fibrous matrices while solvent-loaded agarose hydrogel stamps are used to create microstructures. Compared with other methods, our method shows extreme simplicity and flexibility originated from the mono-/multi-spinneret conversion and limitless micropatterns of agarose hydrogel stamps. Three types of PLLA fibrous matrices including patterned nano-Ag/PLLA hybrid fibers, patterned bicompartment polyethylene terephthalate/PLLA fibers, and patterned hollow PLLA fibers are fabricated and their capability to manipulate biomolecule distribution and functions, that is, bacterial distribution and antibacterial performance, cell patterning and adhesion/spreading behaviors, and protein adsorption and delivery, is demonstrated in detail. The method described in our paper provides a powerful tool to restore spatial complexity in biomimetic matrices and would have promising applications in the field of biomedical engineering.

  20. [Self-assembly tissue engineering fibrocartilage model of goat temporomandibular joint disc].

    Science.gov (United States)

    Kang, Hong; Li, Zhen-Qiang; Bi, Yan-Da

    2011-06-01

    To construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a basis for tissue engineering of the temporomandibular joint disc and other fibrocartilage. Cells from temporomandibular joint discs of goats were harvested and cultured. 5.5 x 10(6) cells were seeded in each agarose well with diameter 5 mm x depth 10 mm, daily replace of medium, cultured for 2 weeks. One day after seeding, goat temporomandibular joint disc cells in agarose wells were gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining was conducted and observed that cells were round and wrapped around by the matrix. Positive Safranin-O/fast green staining for glycosaminoglycans was observed throughout the entire constructs, and picro-sirius red staining was examined and distribution of numerous type I collagen was found. Immunohistochemistry staining demonstrated brown yellow particles in cytoplasm and around extracellular matrix, which showed self-assembly construct can produce type I collagen as native temporomandibular joint disc tissue. Production of extracellular matrix in self-assembly construct as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.

  1. Nanoparticles as image enhancing agents for ultrasonography

    Energy Technology Data Exchange (ETDEWEB)

    Liu Jun [Biomedical Engineering Department, Ohio State University, 270 Bevis Hall, 1080 Carmack Rd, Columbus, OH 43210 (United States); Levine, Andrea L [Department of Veterinary Biosciences, Ohio State University, 1925 Coffey Rd, Columbus, OH 43210 (United States); Mattoon, John S [Department of Veterinary Clinical Sciences, Ohio State University, 1151 Veterinary Hospital, 601 Vernon Tharp St., Columbus, OH 43210 (United States); Yamaguchi, Mamoru [Department of Veterinary Biosciences, Ohio State University, 1925 Coffey Rd, Columbus, OH 43210 (United States); Lee, Robert J [Division of Pharmaceutics, College of Pharmacy, NCI Comprehensive Cancer Center, and NSF Nanoscale Science and Engineering Center, Ohio State University, 500 West 12th Avenue, Columbus, OH 43210 (United States); Pan Xueliang [Department of Statistics, Ohio State University, 1958 Neil Avenue, Columbus, OH 43210 (United States); Rosol, Thomas J [Department of Veterinary Biosciences, Ohio State University, 1925 Coffey Rd, Columbus, OH 43210 (United States)

    2006-05-07

    Nanoparticles have drawn great attention as targeted imaging and/or therapeutic agents. The small size of the nanoparticles allows them to target cells that are beyond capillary vasculature, such as cancer cells. We investigated the effect of solid nanoparticles for enhancing ultrasonic grey scale images in tissue phantoms and mouse livers in vivo. Silica nanospheres (100 nm) were dispersed in agarose at 1-2.5% mass concentration and imaged by a high-resolution ultrasound imaging system (transducer centre frequency: 30 MHz). Polystyrene particles of different sizes (500-3000 nm) and concentrations (0.13-0.75% mass) were similarly dispersed in agarose and imaged. Mice were injected intravenously with nanoparticle suspensions in saline. B-mode images of the livers were acquired at different time points after particle injection. An automated computer program was used to quantify the grey scale changes. Ultrasonic reflections were observed from nanoparticle suspensions in agarose gels. The image brightness, i.e., mean grey scale level, increased with particle size and concentration. The mean grey scale of mouse livers also increased following particle administration. These results indicated that it is feasible to use solid nanoparticles as contrast enhancing agents for ultrasonic imagin000.

  2. Artificial dirt: microfluidic substrates for nematode neurobiology and behavior.

    Science.gov (United States)

    Lockery, S R; Lawton, K J; Doll, J C; Faumont, S; Coulthard, S M; Thiele, T R; Chronis, N; McCormick, K E; Goodman, M B; Pruitt, B L

    2008-06-01

    With a nervous system of only 302 neurons, the free-living nematode Caenorhabditis elegans is a powerful experimental organism for neurobiology. However, the laboratory substrate commonly used in C. elegans research, a planar agarose surface, fails to reflect the complexity of this organism's natural environment, complicates stimulus delivery, and is incompatible with high-resolution optophysiology experiments. Here we present a new class of microfluidic devices for C. elegans neurobiology and behavior: agarose-free, micron-scale chambers and channels that allow the animals to crawl as they would on agarose. One such device mimics a moist soil matrix and facilitates rapid delivery of fluid-borne stimuli. A second device consists of sinusoidal channels that can be used to regulate the waveform and trajectory of crawling worms. Both devices are thin and transparent, rendering them compatible with high-resolution microscope objectives for neuronal imaging and optical recording. Together, the new devices are likely to accelerate studies of the neuronal basis of behavior in C. elegans.

  3. Lymphocyte receptors for pertussis toxin

    Energy Technology Data Exchange (ETDEWEB)

    Clark, C.G.; Armstrong, G.D. (Univ. of Alberta, Edmonton (Canada))

    1990-12-01

    We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

  4. How to assess the plasma delivery of RONS into tissue fluid and tissue

    Science.gov (United States)

    Oh, Jun-Seok; Szili, Endre J.; Gaur, Nishtha; Hong, Sung-Ha; Furuta, Hiroshi; Kurita, Hirofumi; Mizuno, Akira; Hatta, Akimitsu; Short, Robert D.

    2016-08-01

    The efficacy of helium (He) and argon (Ar) plasma jets are being investigated for different healthcare applications including wound and cancer therapy, sterilisation and surface disinfections. Current research points to a potential link between the generation of reactive oxygen and nitrogen species (RONS) and outcomes in a range of biological and medical applications. As new data accrue, further strengthening this link, it becomes important to understand the controlled delivery of RONS into solutions, tissue fluids and tissues. This paper investigates the use of He and Ar plasma jets to deliver three RONS (hydrogen peroxide—H2O2, nitrite—\\text{NO}2- and nitrate—\\text{NO}3- ) and molecular oxygen (O2) directly into deionised (DI) water, or indirectly into DI water through an agarose target. The DI water is used in place of tissue fluid and the agarose target serves as a surrogate of tissue. Direct plasma jet treatments deliver more RONS and O2 than the through-agarose treatments for equivalent treatments times. The former only deliver RONS whilst the plasma jets are ignited; the latter continues to deliver RONS into the DI water long after the plasmas are extinguished. The He plasma jet is more effective at delivering H2O2 and \\text{NO}2- directly into DI water, but the Ar plasma jet is more effective at nitrating the DI water in both direct and through-agarose treatments. DI water directly treated with the plasma jets is deoxygenated, with the He plasma jet purging more O2 than the Ar plasma jet. This effect is known as ‘sparging’. In contrast, for through-agarose treatments both jets oxygenated the DI water. These results indicate that in the context of direct and indirect plasma jet treatments of real tissue fluids and tissue, the choice of process gas (He or Ar) could have a profound effect on the concentrations of RONS and O2. Irrespective of operating gas, sparging of tissue fluid (in an open wound) for long prolonged periods during direct plasma

  5. Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii

    Directory of Open Access Journals (Sweden)

    Marita G. Pereira

    2017-09-01

    Full Text Available Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr, glyoxyl-agarose (GX, MANAE-agarose activated with glutaraldehyde (GA and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr, at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea, cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA/docosahexaenoic acid (DHA ratio than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of

  6. Preparation of open porous polycaprolactone microspheres and their applications as effective cell carriers in hydrogel system

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingchun [Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering (China); Tan, Ke; Ye, Zhaoyang [State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 China (China); Zhang, Yan, E-mail: zhang_yan@ecust.edu.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering (China); Tan, Wensong [State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 China (China); Lang, Meidong, E-mail: mdlang@ecust.edu.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering (China)

    2012-12-01

    Common hydrogel, composed of synthetic polymers or natural polysaccharides could not support the adhesion of anchorage-dependent cells due to the lack of cell affinitive interface and high cell constraint. The use of porous polyester microspheres as cell-carriers and introduction of cell-loaded microspheres into the hydrogel system might overcome the problem. However, the preparation of the open porous microsphere especially using polycaprolactone (PCL) has been rarely reported. Here, the open porous PCL microspheres were fabricated via the combined emulsion/solvent evaporation and particle leaching method. The microspheres exhibited porous surface and inter-connective pore structure. Additionally, the pore structure could be easily controlled by adjusting the processing parameters. The surface pore size could be altered from 20 {mu}m to 80 {mu}m and the internal porosities were varied from 30% to 70%. The obtained microspheres were evaluated to delivery mesenchymal stem cells (MSCs) and showed the improved cell adhesion and growth when compared with the non-porous microspheres. Then, the MSCs loaded microspheres were introduced into agarose hydrogel. MSCs remained alive and sustained proliferation in microsphere/agarose composite in 5-day incubation while a decrement of MSCs viabilities was found in agarose hydrogel without microspheres. The results indicated that the microsphere/hydrogel composite had a great potential in cell therapy and injectable system for tissue regeneration. Highlights: Black-Right-Pointing-Pointer The open porous polycaprolactone microspheres were fabricated using paraffin as a porogen. Black-Right-Pointing-Pointer The microspheres exhibited porous surface and inter-connective pore structure. Black-Right-Pointing-Pointer The surface and internal pore size and porosity of microsphere could be controlled. Black-Right-Pointing-Pointer The porous microspheres exhibited an improved cell adhesion and proliferation. Black

  7. A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.

    Science.gov (United States)

    Vinardell, Tatiana; Sheehy, Eamon J; Buckley, Conor T; Kelly, Daniel J

    2012-06-01

    Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-β3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation

  8. Experimental Assays to Assess the Efficacy of Vinegar and Other Topical First-Aid Approaches on Cubozoan (Alatina alata) Tentacle Firing and Venom Toxicity.

    Science.gov (United States)

    Yanagihara, Angel A; Wilcox, Christie; King, Rebecca; Hurwitz, Kikiana; Castelfranco, Ann M

    2016-01-11

    Despite the medical urgency presented by cubozoan envenomations, ineffective and contradictory first-aid management recommendations persist. A critical barrier to progress has been the lack of readily available and reproducible envenomation assays that (1) recapitulate live-tentacle stings; (2) allow quantitation and imaging of cnidae discharge; (3) allow primary quantitation of venom toxicity; and (4) employ rigorous controls. We report the implementation of an integrated array of three experimental approaches designed to meet the above-stated criteria. Mechanistically overlapping, yet distinct, the three approaches comprised (1) direct application of test solutions on live tentacles (termed tentacle solution assay, or TSA) with single image- and video-microscopy; (2) spontaneous stinging assay using freshly excised tentacles overlaid on substrate of live human red blood cells suspended in agarose (tentacle blood agarose assays, or TBAA); and (3) a "skin" covered adaptation of TBAA (tentacle skin blood agarose assay, or TSBAA). We report the use and results of these assays to evaluate the efficacy of topical first-aid approaches to inhibit tentacle firing and venom activity. TSA results included the potent stimulation of massive cnidae discharge by alcohols but only moderate induction by urine, freshwater, and "cola" (carbonated soft drink). Although vinegar, the 40-year field standard of first aid for the removal of adherent tentacles, completely inhibited cnidae firing in TSA and TSBAA ex vivo models, the most striking inhibition of both tentacle firing and subsequent venom-induced hemolysis was observed using newly-developed proprietary formulations (Sting No More™) containing copper gluconate, magnesium sulfate, and urea.

  9. Analysis of spatial diffusion of ferric ions in PVA-GTA gel dosimeters through magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Marrale, Maurizio [Dipartimento di Fisica e Chimica, Universitá di Palermo, Viale delle Scienze, Edificio 18, 90128 Palermo (Italy); Istituto Nazionale di Fisica Nucleare (INFN) – Gruppo V Sezione di Catania, Via Santa Sofia, 64, 95123 Catania (Italy); ATeN Center, Università di Palermo, Viale delle Scienze, Edificio 18, 90128 Palermo (Italy); Collura, Giorgio [Dipartimento di Fisica e Chimica, Universitá di Palermo, Viale delle Scienze, Edificio 18, 90128 Palermo (Italy); Istituto Nazionale di Fisica Nucleare (INFN) – Gruppo V Sezione di Catania, Via Santa Sofia, 64, 95123 Catania (Italy); Gallo, Salvatore, E-mail: salvatore.gallo05@unipa.it [Dipartimento di Fisica e Chimica, Universitá di Palermo, Viale delle Scienze, Edificio 18, 90128 Palermo (Italy); Istituto Nazionale di Fisica Nucleare (INFN) – Gruppo V Sezione di Catania, Via Santa Sofia, 64, 95123 Catania (Italy); Dipartimento di Fisica, Universitá di Milano, Via Giovanni Celoria 16, 20133 Milano (Italy); Nici, Stefania [Dipartimento di Fisica e Chimica, Universitá di Palermo, Viale delle Scienze, Edificio 18, 90128 Palermo (Italy); Tranchina, Luigi [ATeN Center, Università di Palermo, Viale delle Scienze, Edificio 18, 90128 Palermo (Italy); Abbate, Boris Federico [U.O.C. Fisica Sanitaria, A.R.N.A.S., Ospedale Civico Palermo, Piazza Nicola Leotta 4, 90127 Palermo (Italy); Marineo, Sandra; Caracappa, Santo [Istituto Zooprofilattico Sperimentale della Sicilia (IZS), Via Gino Marinuzzi, 3, 90129 Palermo (Italy); and others

    2017-04-01

    Highlights: • Analysis of ferric ions diffusion throughout the gel matrix in PVA-GTA samples. • Measurements with preclinical 7T MRI scanner with spatial resolution of 200 μm. • Diffusion process is much slower for PVA-GTA gels than for agarose ones. - Abstract: This work focused on the analysis of the temporal diffusion of ferric ions through PVA-GTA gel dosimeters. PVA-GTA gel samples, partly exposed with 6 MV X-rays in order to create an initial steep gradient, were mapped using magnetic resonance imaging on a 7T MRI scanner for small animals. Multiple images of the gels were acquired over several hours after irradiation and were analyzed to quantitatively extract the signal profile. The spatial resolution achieved is 200 μm and this makes this technique particularly suitable for the analysis of steep gradients of ferric ion concentration. The results obtained with PVA-GTA gels were compared with those achieved with agarose gels, which is a standard dosimetric gel formulation. The analysis showed that the diffusion process is much slower (more than five times) for PVA-GTA gels than for agarose ones. Furthermore, it is noteworthy that the diffusion coefficient value obtained through MRI analysis is significantly consistent with that obtained in separate study Marini et al. (Submitted for publication) using a totally independent method such as spectrophotometry. This is a valuable result highlighting that the good dosimetric features of this gel matrix not only can be reproduced but also can be measured through independent experimental techniques based on different physical principles.

  10. An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds.

    Science.gov (United States)

    Zhang, Shaohua; Jiang, Zhongyi; Shi, Jiafu; Wang, Xueyan; Han, Pingping; Qian, Weilun

    2016-09-28

    Design and preparation of high-performance immobilized biocatalysts with exquisite structures and elucidation of their profound structure-performance relationship are highly desired for green and sustainable biotransformation processes. Learning from nature has been recognized as a shortcut to achieve such an impressive goal. Loose connective tissue, which is composed of hierarchically organized cells by extracellular matrix (ECM) and is recognized as an efficient catalytic system to ensure the ordered proceeding of metabolism, may offer an ideal prototype for preparing immobilized biocatalysts with high catalytic activity, recyclability, and stability. Inspired by the hierarchical structure of loose connective tissue, we prepared an immobilized biocatalyst enabled by microcapsules-in-hydrogel (MCH) scaffolds via biomimetic mineralization in agarose hydrogel. In brief, the in situ synthesized hybrid microcapsules encapsulated with glucose oxidase (GOD) are hierarchically organized by the fibrous framework of agarose hydrogel, where the fibers are intercalated into the capsule wall. The as-prepared immobilized biocatalyst shows structure-dependent catalytic performance. The porous hydrogel permits free diffusion of glucose molecules (diffusion coefficient: ∼6 × 10(-6) cm(2) s(-1), close to that in water) and retains the enzyme activity as much as possible after immobilization (initial reaction rate: 1.5 × 10(-2) mM min(-1)). The monolithic macroscale of agarose hydrogel facilitates the easy recycling of the immobilized biocatalyst (only by using tweezers), which contributes to the nonactivity decline during the recycling test. The fiber-intercalating structure elevates the mechanical stability of the in situ synthesized hybrid microcapsules, which inhibits the leaching and enhances the stability of the encapsulated GOD, achieving immobilization efficiency of ∼95%. This study will, therefore, provide a generic method for the hierarchical organization of (bio

  11. Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels

    Directory of Open Access Journals (Sweden)

    JT Connelly

    2011-09-01

    Full Text Available Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10 and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli.

  12. Local Delivery of Fluorescent Dye For Fiber-Optics Confocal Microscopy of the Living Heart

    Directory of Open Access Journals (Sweden)

    Chao eHuang

    2014-09-01

    Full Text Available Fiber-optics confocal microscopy (FCM is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release versus foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  13. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    Science.gov (United States)

    Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  14. A comparison of self-assembly and hydrogel encapsulation as a means to engineer functional cartilaginous grafts using culture expanded chondrocytes.

    Science.gov (United States)

    Mesallati, Tariq; Buckley, Conor T; Kelly, Daniel J

    2014-01-01

    Despite an increased interest in the use of hydrogel encapsulation and cellular self-assembly (often termed "self-aggregating" or "scaffold-free" approaches) for tissue-engineering applications, to the best of our knowledge, no study to date has been undertaken to directly compare both approaches for generating functional cartilaginous grafts. The objective of this study was to directly compare self-assembly (SA) and agarose hydrogel encapsulation (AE) as a means to engineer such grafts using passaged chondrocytes. Agarose hydrogels (5 mm diameter × 1.5 mm thick) were seeded with chondrocytes at two cell seeding densities (900,000 cells or 4 million cells in total per hydrogel), while SA constructs were generated by adding the same number of cells to custom-made molds. Constructs were either supplemented with transforming growth factor (TGF)-β3 for 6 weeks, or only supplemented with TGF-β3 for the first 2 weeks of the 6 week culture period. The SA method was only capable of generating geometrically uniform cartilaginous tissues at high seeding densities (4 million cells). At these high seeding densities, we observed that total sulphated glycosaminoglycan (sGAG) and collagen synthesis was greater with AE than SA, with higher sGAG retention also observed in AE constructs. When normalized to wet weight, however, SA constructs exhibited significantly higher levels of collagen accumulation compared with agarose hydrogels. Furthermore, it was possible to engineer such functionality into these tissues in a shorter timeframe using the SA approach compared with AE. Therefore, while large numbers of chondrocytes are required to engineer cartilaginous grafts using the SA approach, it would appear to lead to the faster generation of a more hyaline-like tissue, with a tissue architecture and a ratio of collagen to sGAG content more closely resembling native articular cartilage.

  15. Systematic random sampling of the comet assay.

    Science.gov (United States)

    McArt, Darragh G; Wasson, Gillian R; McKerr, George; Saetzler, Kurt; Reed, Matt; Howard, C Vyvyan

    2009-07-01

    The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

  16. Radiation effects on human glia and glioma cells in vitro

    International Nuclear Information System (INIS)

    Nilsson, S.

    1983-01-01

    The radiosensitivity of human glia and glioma cells has been studied in vitro, and a new cloning method has been developed to overcome the difficulties due to the very low cloning efficiency of these cells. The cells were confined to small palladium areas surrounded by agarose, which increased the cell density, but kept the clones separated. Using this method, the glia cells were found to be very sensitive to gamma irradiation (D 0 =1.0-1.5 Gy and n=1) in comparision with the glioma cells (D 0 =1.5-2.5 Gy and n=3.5). The induction and repair of DNA strand breaks were studied with two DNA unwinding techniques. No differences between the two cell-lines were detected when induction and fast repair were studied with the single-labelling method, while the glioma cells showed less unrepaired DNA strand breaks than the glia cells after 1, 2 and 3 hours, when the double-labelling method was used. Detachment, attachment and growth kinetics were studied using the palladium-agarose cloning method. All of the glioma cell-lines studied, detached and attached themselves at rates higher than the normal diploid glia cell-lines. All of the cell-lines contained clones with different properties. Some clones were rapidly growing, others maintained a nearly constant number of cells or even decreased. The effects of chronic hypoxia were tested in a few experiments. Low oxygen tension in the culture medium reduced the rate of growth and the DNA synthesis of the glioma cells. The present study indicates that cultured human glioma cells are less radiosensitive than cultured glia cells. The palladium-agarose technique, enable studying growth kinetics detachment, attachment and radiosensitivity in a quantitative manner for cells with low cloning efficiency. (author)

  17. A comparison of different bioinks for 3D bioprinting of fibrocartilage and hyaline cartilage.

    Science.gov (United States)

    Daly, Andrew C; Critchley, Susan E; Rencsok, Emily M; Kelly, Daniel J

    2016-10-07

    Cartilage is a dense connective tissue with limited self-repair capabilities. Mesenchymal stem cell (MSC) laden hydrogels are commonly used for fibrocartilage and articular cartilage tissue engineering, however they typically lack the mechanical integrity for implantation into high load bearing environments. This has led to increased interested in 3D bioprinting of cell laden hydrogel bioinks reinforced with stiffer polymer fibres. The objective of this study was to compare a range of commonly used hydrogel bioinks (agarose, alginate, GelMA and BioINK™) for their printing properties and capacity to support the development of either hyaline cartilage or fibrocartilage in vitro. Each hydrogel was seeded with MSCs, cultured for 28 days in the presence of TGF-β3 and then analysed for markers indicative of differentiation towards either a fibrocartilaginous or hyaline cartilage-like phenotype. Alginate and agarose hydrogels best supported the development of hyaline-like cartilage, as evident by the development of a tissue staining predominantly for type II collagen. In contrast, GelMA and BioINK ™ (a PEGMA based hydrogel) supported the development of a more fibrocartilage-like tissue, as evident by the development of a tissue containing both type I and type II collagen. GelMA demonstrated superior printability, generating structures with greater fidelity, followed by the alginate and agarose bioinks. High levels of MSC viability were observed in all bioinks post-printing (∼80%). Finally we demonstrate that it is possible to engineer mechanically reinforced hydrogels with high cell viability by co-depositing a hydrogel bioink with polycaprolactone filaments, generating composites with bulk compressive moduli comparable to articular cartilage. This study demonstrates the importance of the choice of bioink when bioprinting different cartilaginous tissues for musculoskeletal applications.

  18. DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis

    International Nuclear Information System (INIS)

    Wiesehahn, G.; Hearst, J.E.

    1978-01-01

    Derivatives of furo[3,2-g]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength uv light, the bound psoralen may react covalently with pyrimidine residues on one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 28 0 +- 4 0 . For 4,5',8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, we calculated an unwinding angle of 29 0 +- 3 0 . Assuming an unwinding angle of 28 0 for the noncovalent intercalation of psoralen derivatives, we used the same procedure to determine intercalation binding constants. The association constants for 4'-aminomethyltrioxsalen were 300 to 1400 M -1 in NaCl at 0.2 to 0.05 M and 300 to 2500 M -1 in Mg 2+ at 4 to 0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg 2+ was determined to be 70 M -1

  19. Apolipoprotein A-I metabolism in cynomolgus monkey. Identification and characterization of beta-migrating pools

    International Nuclear Information System (INIS)

    Melchior, G.W.; Castle, C.K.

    1989-01-01

    Fresh plasma from control (C) and hypercholesterolemic (HC) cynomolgus monkeys was analyzed by agarose electrophoresis-immunoblotting with antibody to cynomolgus monkey apolipoprotein (apo) A-I. Two bands were evident on the autoradiogram: an alpha-migrating band (high density lipoprotein) and a beta-migrating band that comigrated exactly with cynomolgus monkey low density lipoprotein (LDL). The presence of beta-migrating apo A-I in the plasma of these monkeys was confirmed by Geon-Pevikon preparative electrophoresis, crossed immunoelectrophoresis, and isotope dilution studies in which radiolabeled apo A-I was found to equilibrate also with alpha- and beta-migrating pools of apo A-I in the plasma. Subfractionation of C and HC plasma by agarose column chromatography (Bio-Gel A-0.5M and A-15M) followed by agarose electrophoresis-immunoblotting indicated that the beta-migrating apo A-I in C was relatively homogeneous and eluted with proteins of Mr approximately 50 kD [apo A-I(50 kD)], whereas two beta-migrating fractions were identified in HC, one that eluted with the 50-kD proteins, and the other that eluted in the LDL Mr range [apo A-I(LDL)]. The apo A-I(LDL) was precipitated by antibody to cynomolgus monkey apo B. The apo A-I(50 kD) accounted for 5 +/- 1% (mean +/- SD) of the plasma apo A-I in C plasma, and 15 +/- 7% in HC plasma. No apo A-I(LDL) was detected in C plasma, but that fraction accounted for 9 +/- 7% of the apo A-I in HC plasma. These data establish the presence of multiple pools of apo A-I in the cynomolgus monkey, which must be taken into consideration in any comprehensive model of apo A-I metabolism in this species

  20. A sperm-agglutinating lectin from seeds of Jack fruit (Artocarpus heterophyllus).

    Science.gov (United States)

    Namjuntra, P; Muanwongyathi, P; Chulavatnatol, M

    1985-04-30

    A lectin specific for N-acetylgalactosamine was isolated from seed extract of Jack fruit (Artocarpus heterophyllus) by ammonium sulfate precipitation, followed by affinity chromatography on a Affigel-galactosamine-agarose column. The lectin possessed agglutinating activities for human and rat sperm as well as human red blood cells. It was found to have Mr = 62,000 consisting of two dissimilar subunits of Mr = 18,000 and 13,000. It also cross-reacted with an antibody against the lectin of Osage Orange (Maclura pomifera).

  1. Reversed phase parallel artificial membrane permeation assay for log P measurement

    Directory of Open Access Journals (Sweden)

    Zihao Song

    2016-03-01

    Full Text Available A reversed phase parallel artificial membrane permeation assay (RP-PAMPA was newly invented for log P measurement. An oil/water/oil sandwich was constructed using a conventional PAMPA instrument. 1 % agarose was used to improve the physical stability of the water phase. A linear correlation between log P and the apparent permeability was observed in the -0.24 < log P < 2.85 region (R2 = 0.98. RP-PAMPA was also applied to pKa measurement.

  2. Effect of gamma radiation on the leakage of substances from lymphocytes. In vitro study using immuno-precipitation techniques in gel medium

    International Nuclear Information System (INIS)

    Dubos, M.; Nguyen, T.-L.; Drouet, J.; Goujon, P.

    In vitro effect of a 5000 R irradiation on rat blood lymphocytes was studied during several days in surviving cell suspensions, at various incubation temperature. Immunochemical analysis in agarose gel, using the simple diffusion technique, exhibited the leakage of two groups of antigenic compounds, from lymphocytes. The first group compounds seemed to be periodical renewal products of surface cell membrane elements, common to lymphocytes and erythrocytes; irradiation increased their release. A correlation was established between the second group compounds and cell metabolism and these compounds seemed to be enzymes of cytolitic origin [fr

  3. Chloramine-T induced binding of monoclonal antibody B72. 3 to concanavalin-A

    Energy Technology Data Exchange (ETDEWEB)

    Cole, W.C.; Jhingran, S.G. (Methodist Hospital, Houston, TX (United States) Baylor Coll. of Medicine, Houston, TX (United States))

    1993-07-01

    The effects of chloramine-T (CT) on monoclonal antibody B72.3 were studied with particular reference to Con-A lectin binding. After exposure to chloramine-T concentrations from 0.8 to 4.0 mg/mL (115-574 mol CT/mol B72.3), B72.3 showed progressive binding to agarose-linked Con-A. This behavior was paralleled by decreasing immunoreactivity and increasing fragmentation and aggregation of B72.3 demonstrated by SDS-PAGE and size exclusion HPLC. (Author).

  4. Isolation and purification of wheat germ agglutinin and analysis of its properties

    Science.gov (United States)

    Wang, Han

    2017-12-01

    In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

  5. Detection of genetically modified rice: Collaborative validation study of a PCR based detection of genetically modified rice Oryza sativa commercially available in Saudi Arabia

    OpenAIRE

    Ibrahim, Mohamed; Alaraidh, Ibrahim; Amid, Azura; Farouk, Abd-El Aziem; Bazaid, Salih; Greiner, Ralf; Alghunaim, Abdullah

    2011-01-01

    A collaborative trial study has been conducted for validation of an extraction method and a subsequent PCR for  the detection of transgenic rice sold in Saudi Arabia. The tests were carried out in Saudi Arabia using Real-Time PCR and the positive samples were validated in another lab in Malaysia using PCR and agarose gel visualization.  The samples were tested for the existence of the NOS Terminator. A total of 150 samples were tested out of which three samples tested positi...

  6. Neutron scattering studies of the dynamics of biopolymer-water systems using pulsed-source spectrometers

    Energy Technology Data Exchange (ETDEWEB)

    Middendorf, H.D. [Univ. of Oxford (United Kingdom); Miller, A. [Stirling Univ., Stirling (United Kingdom)

    1994-12-31

    Energy-resolving neutron scattering techniques provide spatiotemporal data suitable for testing and refining analytical models or computer simulations of a variety of dynamical processes in biomolecular systems. This paper reviews experimental work on hydrated biopolymers at ISIS, the UK Pulsed Neutron Facility. Following an outline of basic concepts and a summary of the new instrumental capabilities, the progress made is illustrated by results from recent experiments in two areas: quasi- elastic scattering from highly hydrated polysaccharide gels (agarose and hyaluronate), and inelastic scattering from vibrational modes of slightly hydrated collagen fibers.

  7. Multilayer mounting for long-term light sheet microscopy of zebrafish.

    Science.gov (United States)

    Weber, Michael; Mickoleit, Michaela; Huisken, Jan

    2014-02-27

    Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

  8. Binding assays with streptavidin-functionalized superparamagnetic nanoparticles and biotinylated analytes using fluxgate magnetorelaxometry

    International Nuclear Information System (INIS)

    Heim, Erik; Ludwig, Frank; Schilling, Meinhard

    2009-01-01

    Binding assays based on the magnetorelaxation of superparamagnetic nanoparticles as markers are presented utilizing a differential fluxgate system. As ligand and receptor, streptavidin and biotin, respectively, are used. Superparamagnetic nanoparticles are functionalized with streptavidin and bound to two types of biotinylated analytes: agarose beads and bovine serum (BSA) proteins. The size difference of the two analytes causes a different progress of the reaction. As a consequence, the analysis of the relaxation signal is carried out dissimilarly for the two analytes. In addition, we studied the reaction kinetics of the two kinds of analytes with the fluxgate system.

  9. Genotoxic and Cytotoxic Safety Evaluation of Papain (Carica papaya L. Using In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Claudia R. da Silva

    2010-01-01

    This work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced-H2O2 oxidative stress in Escherichia coli strains. Cytotoxicity assay, Growth inhibition test, WP2-Mutoxitest and Plasmid-DNA treatment, and agarose gel electrophoresis were used to investigate if papain would present any toxic or mutagenic potential as well as if papain would display antioxidant properties. Papain exhibited negative results for all tests. This agent presented an activity protecting cells against H2O2-induced mutagenesis.

  10. Continuous production of chitooligosaccharides by an immobilized enzyme in a dual-reactor system

    DEFF Research Database (Denmark)

    Santos-Moriano, Paloma; Woodley, John; Plou, Francisco J.

    2016-01-01

    A chitosanolytic activity found in a commercial α-amylase from Bacillus amylolyquefaciens (BAN) was covalently immobilized onto glyoxal agarose beads (25% recovery of activity) and assessed for the continuous production of chitooligosaccharides (COS). The immobilization did not change the reactio......, the productivity of the PBR at the lowest dilution rate was 37 gCOS L−1 h−1, with a conversion yield of 73%. In contrast, at the highest dilution rate, the productivity was nearly 200 gCOS L−1 h−1, but the conversion yield dropped to around 40%....

  11. Identification of Forensic Samples via Mitochondrial DNA in the Undergraduate Biochemistry Laboratory

    Science.gov (United States)

    Millard, Julie T.; Pilon, André M.

    2003-04-01

    A recent forensic approach for identification of unknown biological samples is mitochondrial DNA (mtDNA) sequencing. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify a 440 base pair hypervariable region of human mtDNA from a variety of "crime scene" samples (e.g., teeth, hair, nails, cigarettes, envelope flaps, toothbrushes, and chewing gum). Amplification is verified via agarose gel electrophoresis and then samples are subjected to cycle sequencing. Sequence alignments are made via the program CLUSTAL W, allowing students to compare samples and solve the "crime."

  12. The fractionation of t-RNA on N,N′-bis(3-aminopropyl)-piperazine substituted-Sepharose

    Science.gov (United States)

    Leberman, Reuben; Giovanelli, Ruth; Acosta, Zenobio

    1974-01-01

    An anion exchange agarose has been prepared by modifying sepharose 6B with N,N′-bis (-3-aminopropyl) piperazine. This material (BAPP-Sepharose) has been used for the fractionation of t-RNA from E.coli by column chromatography. The results obtained with gram quantities of crude t-RNA at pH 4.6 and pH 8.0 as measured by the elution patterns of alanyl, arginyl, aspartyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl, tyrosyl, and valyl t-RNA are described. PMID:10793731

  13. Undergraduate physics laboratory: Electrophoresis in chromatography paper

    Science.gov (United States)

    Hyde, Alexander; Batishchev, Oleg

    2015-12-01

    An experiment studying the physical principles of electrophoresis in liquids was developed for an undergraduate laboratory. We have improved upon the standard agarose gel electrophoresis experimental regime with a straightforward and cost-effective procedure, in which drops of widely available black food coloring were separated by electric field into their dye components on strips of chromatography paper soaked in a baking soda/water solution. Terminal velocities of seven student-safe dyes were measured as a function of the electric potential applied along the strips. The molecular mobility was introduced and calculated by analyzing data for a single dye. Sources of systematic and random errors were investigated.

  14. Resonance Energy Transfer between protein and rhamnolipid capped ZnS quantum dots: Application in in-gel staining of proteins

    Science.gov (United States)

    Janakiraman, Narayanan; Mohan, Abhilash; Kannan, Ashwin; Pennathur, Gautam

    The interaction of proteins with quantum dots is an interesting field of research. These interactions occur at the nanoscale. We have probed the interaction of Bovine Serum Albumin (BSA) and Candida rugosa lipase (CRL) with rhamnolipid capped ZnS (RhlZnSQDs) using absorption and fluorescence spectroscopy. Optical studies on mixtures of RhlZnSQDs and proteins resulted in Förster's Resonance Energy Transfer (FRET) from proteins to QDs. This phenomenon has been exploited to detect proteins in agarose gel electrophoresis. The activity of the CRL was unaffected on the addition of QDs as revealed by zymography.

  15. Dynamics of a Camphoric Acid boat at the air-water interface

    OpenAIRE

    Akella, V. S.; Singh, D. K.; Mandre, S.; Bandi, M. M.

    2017-01-01

    We report experiments on an agarose gel tablet loaded with camphoric acid (c-boat) set into self-motion by interfacial tension gradients at the air-water interface. We observe three distinct modes of c-boat motion: harmonic mode where the c-boat speed oscillates sinusoidally in time, a steady mode where the c-boat maintains constant speed, and a relaxation oscillation mode where the c-boat maintains near-zero speed between sudden jumps in speed and position at regular time intervals. Whereas ...

  16. Soroepidemiologia da brucelose canina causada por Brucella canis e Brucella abortus na cidade de Alfenas, MG Seroepidemiology of canine brucellosis caused by Brucella canis and Brucella abortus in Alfenas, MG, Brazil

    Directory of Open Access Journals (Sweden)

    A.C. Almeida

    2004-04-01

    Full Text Available The prevalence of canine brucellosis was evaluated in the city of Alfenas, MG through the technique of agarose gel imunodifusion for Brucella canis and slow serum agglutination test with 2-mercaptoetanol for Brucella abortus. The prevalence was of 14.2% and 2.8%, respectively, for B. canis and B. abortus. The positives, characterized by animals above one year of age (77.8%, and mongrel dogs (56.2%, showed a prevalence of 50 and 48% for males and females, respectively. The canine brucellosis was prevalent in the city principally in dogs of outskirts.

  17. Genotyping of the 19-bp insertion/deletion polymorphism in the 5' flank of beta-hydroxylase gene by dissociation analysis of allele-specific PCR products

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Werge, Thomas

    2005-01-01

    The 19-bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase (DBH) gene has been associated with psychiatric disorders. We have developed a simple, reliable and inexpensive closed-tube assay for genotyping of this polymorphism based upon T(m) determination of amplified...... and a conventional approach based upon agarose gel electrophoresis of amplified fragments revealed complete concordance between the two procedures. The insights obtained in this study may be utilized to develop assays based upon dissociation analysis of PCR products for genotyping of other insertion...

  18. Progress in Development of Improved Ion-Channel Biosensors

    Science.gov (United States)

    Nadeau, Jay L.; White, Victor E.; Maurer, Joshua A.; Dougherty, Dennis A.

    2008-01-01

    Further improvements have recently been made in the development of the devices described in Improved Ion-Channel Biosensors (NPO-30710), NASA Tech Briefs, Vol. 28, No. 10 (October 2004), page 30. As discussed in more detail in that article, these sensors offer advantages of greater stability, greater lifetime, and individual electrical addressability, relative to prior ion-channel biosensors. In order to give meaning to a brief description of the recent improvements, it is necessary to recapitulate a substantial portion of the text of the cited previous article. The figure depicts one sensor that incorporates the recent improvements, and can be helpful in understanding the recapitulated text, which follows: These sensors are microfabricated from silicon and other materials compatible with silicon. Typically, the sensors are fabricated in arrays in silicon wafers on glass plates. Each sensor in the array can be individually electrically addressed, without interference with its neighbors. Each sensor includes a well covered by a thin layer of silicon nitride, in which is made a pinhole for the formation of a lipid bilayer membrane. In one stage of fabrication, the lower half of the well is filled with agarose, which is allowed to harden. Then the upper half of the well is filled with a liquid electrolyte (which thereafter remains liquid) and a lipid bilayer is painted over the pinhole. The liquid contains a protein that forms an ion channel on top of the hardened agarose. The combination of enclosure in the well and support by the hardened agarose provides the stability needed to keep the membrane functional for times as long as days or even weeks. An electrode above the well, another electrode below the well, and all the materials between the electrodes together constitute a capacitor. What is measured is the capacitive transient current in response to an applied voltage pulse. One notable feature of this sensor, in comparison with prior such sensors, is a

  19. The use of AMPPD as an alternative substrate for AP-mediated detection of nonradiolabeled DNA probes in Eucalyptus saligna

    OpenAIRE

    De Moura Campos Pardini, M. I. [UNESP; Wolff, J. L C [UNESP; Lopes, C. R. [UNESP

    1993-01-01

    We present a non-radioactive alternative to Southern's (J. Mol. Biol. 98: 503-517, 1975) DNA-DNA hybridization technique. The use of AMPPD - Disodium 3-(4-Methoxyspiro {1,2-dioxetane-3,2'tricyclo[3.3.1.1(3,7)]decan}-4-yl)phyenyl phosphate as an alternative substrate for AP-mediated detection of digoxigenin-11 dUTP-labeled probes made possible the simple and nonhazardous reuse of blots. We used 0.8 % agarose gels containing 30 mug per lane of Eucalyptus saligna DNA, digested with Eco RI, elect...

  20. Study of DNA damage with a new system for irradiation of samples in a nuclear reactor

    Energy Technology Data Exchange (ETDEWEB)

    Gual, Maritza R., E-mail: mrgual@instec.c [Instituto Superior de Tecnologias y Ciencias Aplicadas, InSTEC, Avenida Salvador Allende y Luaces, Quinta de Los Molinos, Plaza de la Revolucion, Havana, AP 6163 (Cuba); Milian, Felix M. [Universidade Estadual de Santa Cruz, UESC (Brazil); Deppman, Airton [Instituto de Fisica, Universidad de Sao Paulo, IF-USP, Rua do Matao, Travessa R, no. 187, Ciudade Universitaria, Butanta, CEP 05508-900, Sao Paulo (Brazil); Coelho, Paulo R.P. [Instituto de Pesquisas Energeticas e Nucleares, IPEN-CNEN/SP (Brazil)

    2011-02-15

    In this paper, we report results of a quantitative analysis of the effects of neutrons on DNA, and, specifically, the production of simple and double breaks of plasmid DNA in aqueous solutions with different concentrations of free-radical scavengers. The radiation damage to DNA was evaluated by electrophoresis through agarose gels. The neutron and gamma doses were measured separately with thermoluminescent detectors. In this work, we have also demonstrated usefulness of a new system for positioning and removing samples in channel BH3 of the IEA-R1 reactor at the Instituto de Pesquisas Energeticas e Nucleares (Brazil) without necessity of interrupting the reactor operation.

  1. Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

    Science.gov (United States)

    Ostberg, C.O.; Rodriguez, R.J.

    2002-01-01

    A suite of 26 PCR-based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species-specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.

  2. OBTENÇÃO DE PLANTAS DE LIMÃO CRAVO (Citrus limonia Osbeck) E TANGERINA CLEÓPATRA (Citrus reshni Hort.) A PARTIR DO CULTIVO DE PROTOPLASTOS DE SUSPENSÃO CELULAR PLANT REGENERATION OF 'RANGPUR' LIME (Citrus limonia Osbeck) AND 'CLEÓPATRA' MANDARIN (Citrus reshni Hort.) THROUGH PROTOPLASTS OF CELL SUSPENSION

    OpenAIRE

    Rodrigo Rocha Latado; Fernando Berlink D'utra Vaz; Augusto Tulmann Neto

    1999-01-01

    Este trabalho descreve uma metodologia para a regeneração de plantas de tangerina 'Cleópatra' e limão 'Cravo', a partir do cultivo de protoplastos de suspensão celular. Para tal, calos nucelares foram induzidos em meio contendo BAP e cultivados em meio sem reguladores de crescimento. Protoplastos foram isolados de suspensões celulares e cultivados em gotas de agarose, com densidade de 2 X 105 protoplastos.ml-1. O meio MT, contendo ácido giberélico e água de coco, foi eficiente na germinação d...

  3. Biodiversity of Thermophilic Prokaryotes with Hydrolytic Activities in Hot Springs of Uzon Caldera, Kamchatka (Russia)▿ †

    OpenAIRE

    Kublanov, Ilya V.; Perevalova, Anna A.; Slobodkina, Galina B.; Lebedinsky, Aleksander V.; Bidzhieva, Salima K.; Kolganova, Tatyana V.; Kaliberda, Elena N.; Rumsh, Lev D.; Haertlé, Thomas; Bonch-Osmolovskaya, Elizaveta A.

    2008-01-01

    Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87°C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or α- or β-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric su...

  4. Low cost, low tech SNP genotyping tools for resource-limited areas: Plague in Madagascar as a model.

    Directory of Open Access Journals (Sweden)

    Cedar L Mitchell

    2017-12-01

    Full Text Available Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs, can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs.To demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar, we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM. We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort.The success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable

  5. Crossed radio-immunoisoelectric focusing as a method of identifying isoallergens: identification of isoallergens in rat urine extracts

    International Nuclear Information System (INIS)

    Longbottom, J.L.

    1984-01-01

    A method of 2-dimensional radio-immunoelectrophoresis to detect directly the presence of 'isoallergens' in complex allergenic (skin test) extracts is described. This procedure, in which the components are separated by isoelectric focusing in agarose gel in the first dimension is therefore basically similar to that of crossed radio-immunoelectrophoresis, and hence has been termed crossed radio-immunoisoelectric focusing. The method has been applied to the allergens present in rat urine and has verified the presence of the cross-reacting α 2 -euglobulin and prealbumin components in (at least) 3 and 2 isoallergenic forms respectively. (Auth.)

  6. Observation of microorganism colonies using a scanning-laser-beam pH-sensing microscope

    International Nuclear Information System (INIS)

    Nakao, M.; Inoue, S.; Oishi, R.; Yoshinobu, T.; Iwasaki, H.

    1995-01-01

    The extracellular pH-distribution of colonies of Saccharomyces cerevisiae (yeast) and Escherichia coli (E. coli) were observed using a newly-developed scanning-laser-beam pH-sensing microscope. Colonies were incubated either on top of agarose plates or between the pH-sensing surface and the agar. In the latter case, colony growth was observed in-situ. The colonies could be observed within a period as short as 8 h for E. coli. The pH-distribution profiles by the colonies were found to be very sharp, in agreement with simulation results. (author)

  7. Isotope separation

    International Nuclear Information System (INIS)

    Rosevear, A.; Sims, H.E.

    1985-01-01

    sup(195m)Au for medical usage is separated from sup(195m)Hg in a solution containing ions of sup(195m)Hg by contacting the solution with an adsorbing agent to adsorb 195 Hgsup(H) thereon, followed by selective elution of sup(195m)Au generated by radioactive decay of the sup(195m)Hg. The adsorbing agent comprises a composite material in the form of an inert porous inorganic substrate (e.g. Kieselguhr),the pores of which are occupied by a hydrogel of a polysaccharide (e.g. agarose) carrying terminal thiol groups for binding Hgsup(H) ions. (author)

  8. Measurement of thermal properties of magnetic nanoparticles using infrared thermal microscopy

    DEFF Research Database (Denmark)

    Kim, Jae Young; Chang, Ki Soo; Kook, Myung Ho

    2013-01-01

    Magnetic nanoparticles (MNPs) are considered promising for biomedical applications such as hyperthermia treatment and disease diagnosis owing to their distinctive thermal properties. For these applications, it is essential to screen the temperature distribution in the targeted disease site....... This study aimed to investigate and observe the thermal properties of a small amount of MNPs used as highly sensitive biomarkers for disease diagnosis by microthermography. Toward this end, we used polyacrylamide and agarose phantoms containing a small amount of MNPs (30 mg Fe-1). In phantoms, the increasing...

  9. Identification of E. coli O157:H7 by Using Specific Primers for rfbE and stx2b Genes

    Directory of Open Access Journals (Sweden)

    Mostafa Bakhshi

    2017-07-01

    Sorbitol-MacConkey agar was used to verification of growth ability of selected colonies during PCR. Results: By appearance of the bonds belong to rfbE and stx2B genes on agarose gel, the ability of designed primers in gene detection in samples of E .coli O157:H7 was verified. Colonies which selected during PCR have growth potency on sorbitol-MacConkey agar medium. Conclusion: It was revealed that we can prepare a fast, precise and relative comfortable method for detection of E. coli O157:H7 strain by using PCR technique and specific primers than other available methods.

  10. Directing functional chemistries on micropatterned conducting polymers for all-polymer cell analysis microsystems

    DEFF Research Database (Denmark)

    Lind, Johan Ulrik; Daugaard, Anders Egede; Andresen, Thomas Lars

    Micrometer scale electrical circuits of PEDOT (poly(3,4-dioxythiophene)) were created by locally oxidizing PEDOT thin films with an agarose stamp containing the oxidizing agent NaOCl. The oxidized PEDOT was removed completely by applying detergents. The process was sufficiently mild that chemical...... groups on the underlying substrate, such as azides or alkynes, were preserved for subsequent specific functionalization. Moreover entire PMOXA (poly(2-methyl-2-oxazoline)) films preventing cell binding could be hidden below the PEDOT and be re-exposed upon stamping, allowing for cell capturing...... microelectrodes on a cell non-adhesive background. Chemically functionalized PEDOT types permitted the introduction of multiple additional types of micropatterned chemistry....

  11. DNA-directed self-assembly of gold nanoparticles into binary and ternary nanostructures

    International Nuclear Information System (INIS)

    Yao Hui; Yi Changqing; Tzang Chihung; Zhu Junjie; Yang Mengsu

    2007-01-01

    The assembly and characterization of gold nanoparticle-based binary and ternary structures are reported. Two strategies were used to assemble gold nanoparticles into ordered nanoscale architectures: in strategy 1, gold nanoparticles were functionalized with single-strand DNA (ssDNA) first, and then hybridized with complementary ssDNA-labelled nanoparticles to assemble designed architectures. In strategy 2, the designed architectures were constructed through hybridization between complementary ssDNA first, then by assembling gold nanoparticles to the scaffolding through gold-sulfur bonds. Both TEM measurements and agarose gel electrophoresis confirmed that the latter strategy is more efficient in generating the designed nanostructures

  12. Antibacterial Compounds from Red Seaweeds (Rhodophyta

    Directory of Open Access Journals (Sweden)

    Noer Kasanah

    2015-07-01

    Full Text Available Seaweeds produce great variety of metabolites benefit for human. Red seaweeds (Rhodophyta are well known as producer of phycocolloids such agar, agarose, carragenan and great variety of secondary metabolites. This review discusses the red algal secondary metabolites with antibacterial activity. The chemical constituents of red algae are steroid, terpenoid, acetogenin and dominated by halogenated compounds mainly brominated compounds. Novel compounds with intriguing skeleton are also reported such as bromophycolides and neurymenolides. In summary, red seaweeds are potential sources for antibacterial agents and can serve as lead in synthesis of new natural medicines.

  13. SU-G-IeP4-08: Initial Investigations of Up-Converting Nanoparticles (UCNP) for 3D Tissue Imaging in Optical-ECT

    International Nuclear Information System (INIS)

    Yoon, S; Dewhirst, M; Oldham, M; Langloss, B; Boss, M; Birer, S

    2016-01-01

    Purpose: Near-IR absorptive up-converting nanoparticles (UCNPs) is a novel contrast for optical-ECT that allows auto-fluorescence-free 3D imaging of labeled cells in a matrix of large (∼1cm 3 ) unsectioned normal tissue. This has the potential to image small metastases or dormant cells that is difficult with down-converting fluorescing dyes due to auto-fluorescence. The feasibility of imaging UCNP in agarose phantoms and a mouse lung is demonstrated, aided by a 3D-printed optical-ECT stage designed to excite UCNP in a mouse lung. Methods: The UCNP, NaYF 4 :Yb/Er (20/2%), studied in this work up-converts 980nm light to visible light peaking sharply at ∼540nm. To characterize the UCNP emission as a function of UCNP concentration, cylindrical 2.5%wt agarose phantoms infused with UCNP at concentrations of 25µg/mL and 50µg/mL were exposed to 1.5W 980nm laser coupled to an optical fiber. The fiber was held stably at 1cm above the stage via a custom 3D-printed stage. An optically cleared lung harvested from a BALBc mice was then injected with 100µL of 1mg/mL UCNP solution ex vivo. Tomographic imaging of the UCNP emission in lung was performed. Results: The laser beam tract is visualized within the agarose phantom. A line profile of UCNP emission at 25µg/mL versus 50µg/mL shows that increasing the UCNP concentration increases emission count. UCNPs injected into a cleared mouse lung disperse throughout the respiratory tract, allowing for visualization and 3D reconstruction. Excitation before and after UCNP injection shows the tissue exhibits no auto-fluorescence at 980nm, allowing clear view of the UCNP without any obscuring features such as conventional down-converting fluorescent tags. Conclusion: We confirm that up-conversion in tissue circumvents completely tissue auto-fluorescence, which allowed background-free 3D reconstruction of the UCNP distribution. We also confirm that raising the UCNP concentration increases emission and that UCNPs are retained in

  14. Rapid detection of genetically diverse tomato black ring virus isolates using reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Hasiów-Jaroszewska, Beata; Budzyńska, Daria; Borodynko, Natasza; Pospieszny, Henryk

    2015-12-01

    A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV.

  15. Mechanism and Regulation of Gene Expression by Androgen Receptor in Prostate Cancer

    Science.gov (United States)

    2004-07-01

    sense and antisense) were generated by in vitro transcription with T7 and acetic acid (NTA)-agarose in the presence of 6 M urea (8). TFIIAa-y was T3 RNA...R1 3 2 RPB6 --* 1 o TRAP95--+ " AES - + + - - + + HDACI -*g TSA -- - - + + + + HDA 3-*R1881 + - + - + - + - 123 SRB7 MED7 U1 UI<, HP IP 0 0 MED7...TLE proteins show poor sequence conservation AR-dependent transcription in vivo, although the exact mech- (at the amino acid level) in both

  16. Microbial community structure of surface sediments from a tropical estuarine environment using next generation sequencing

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, L.; Kuchi, N.; Kale, D.; Anil, A.C.

    are of nearly identical length (~50 km each), are highly productive and dynamic systems and have wide mouth regions and longer flushing periods. In this study, Chicalim (15°24'10.92"N, 73°51'8.55"E) and Siridao (15°25'41.89"N, 73°52'38.84"E) were sampled..., Australia). The DNA was extracted using bead beating and column purification which was performed according to the manufacturer's guidelines. The metagenomic DNA was quantified by Eppendorf-Biospectrometer, and run on 0.8% agarose gel. The gel was viewed...

  17. 1-Alpha Hydroxyvitamin D(5) as a Chemotherapeutic and Possibly Chemopreventive Agent

    Science.gov (United States)

    2007-03-01

    GTT GCT GTT TGT TTG AC, and the antisense primer was 50-CTT CTG TGA GGC TGT TTT TG. The primer for the housekeeping gene G3PDH was purchased from...reverse-transcribed. The cDNA was ampli- fied using Taq polymerase and separated on 1.5% agarose gel. As shown in Fig. 5, the housekeeping gene G3PDH (C...control housekeeping gene. 784 G. Lazzaro et al. / European Journal of Cancer 36 (2000) 780±786 Appendix 3. Publications DAMD17-99-1-9223 – Final

  18. Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

    OpenAIRE

    Sundseth, S S; Nix, C E; Waters, L C

    1990-01-01

    Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were e...

  19. Enriched reaction center preparation from green photosynthetic bacteria. [Chlorobium limicola

    Energy Technology Data Exchange (ETDEWEB)

    Olson, J M; Giddings, Jr, T H; Shaw, E K

    1976-01-01

    Bacteriochlorophyll a reaction-center complex I from Chlorobium limicola f. thiosulfatophilum 6230 (Tassajara) was incubated in 2 M guanidine . HCl and then chromatographed on cross-linked dextran or agarose gel. Two principal components were separated: a larger component with photochemical activity (bacteriochlorophyll a reaction-center complex II) and a smaller component without activity (bacteriochlorophyll a protein). Complex II contains carotenoid, bacteriochlorophyll a, reaction center(s), and cytochromes b and c, but lacks the well characterized bacteriochlorophyll a protein contained in Complex I. Complex II carries out a light-induced reduction of cytochrome b along with an oxidation of cytochrome c.

  20. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    Directory of Open Access Journals (Sweden)

    José Carlos Pelielo de Mattos

    2008-12-01

    Full Text Available Reactive oxygen species (ROS can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2 is a ROS generator, leading to lethality in Escherichia coli (E. coli, with the base excision repair (BER mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.Espécies reativas de oxigênio (ERO podem induzir lesões em diferentes alvos celulares, incluindo o DNA. O cloreto estanoso (SnCl2 é um gerador de ERO que induz letalidade em E. coli, sendo o reparo por excisão de bases (BER um mecanismo importante neste processo. Técnicas como o ensaio cometa (em eucariotos e a eletroforese de DNA plasmidial em gel de agarose têm sido utilizadas para detectar genotoxicidade. No presente estudo, uma adaptação do método de eletroforese em gel alcalino de agarose foi usada para verificar a indução de quebras, pelo SnCl2, no DNA de E. coli, bem como a participação de enzimas do BER na restauração das lesões. Os resultados mostraram que o SnCl2 induziu quebras no DNA de todas as cepas testadas. Além disso, endonuclease IV e exonuclease III estão envolvidas na reparação dos danos. Em resumo, os dados obtidos indicam que a metodologia de eletroforese em gel alcalino de agarose pode ser empregada tanto para o estudo de quebras no DNA, quanto para avaliação dos

  1. Neutron scattering studies of the dynamics of biopolymer-water systems using pulsed-source spectrometers

    International Nuclear Information System (INIS)

    Middendorf, H.D.; Miller, A.

    1994-01-01

    Energy-resolving neutron scattering techniques provide spatiotemporal data suitable for testing and refining analytical models or computer simulations of a variety of dynamical processes in biomolecular systems. This paper reviews experimental work on hydrated biopolymers at ISIS, the UK Pulsed Neutron Facility. Following an outline of basic concepts and a summary of the new instrumental capabilities, the progress made is illustrated by results from recent experiments in two areas: quasi- elastic scattering from highly hydrated polysaccharide gels (agarose and hyaluronate), and inelastic scattering from vibrational modes of slightly hydrated collagen fibers

  2. Pyrimidine dimer formation and repair in human skin

    International Nuclear Information System (INIS)

    Sutherland, B.M.; Harber, L.C.; Kochevar, I.E.

    1980-01-01

    Cyclobutyl pyrimidine dimers have been detected in the DNA of human skin following in vivo irradiation with suberythermal doses of ultraviolet (UV) radiation from FS-20 sun lamp fluorescent tubes. Dimers were assayed by treatment of extracted DNA with Micrococus luteus UV-specific endonuclease, alkaline agarose electrophoresis, and ethidum bromide staining. This technique, in contrast to conventional dimer assays, can be used with nonradioactive DNA and is optimal at low UV light doses. These data suggest that some dimer disappearance by excision repair occurs within 20 min of UV irradiation and that photoreactivation of dimers can make a contribution to the total repair process

  3. Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

    Science.gov (United States)

    Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

    2014-05-01

    The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

  4. Detection of hepatitis C virus RNA using reverse transcription PCR

    International Nuclear Information System (INIS)

    Yap, S.F.

    1998-01-01

    Detection of the viral genome (HCV RNA) is by a combination of cDNA synthesis and PCR followed by gel analysis and/or hybridization assay. In principle, cDNA is synthesized using the viral RNA as template and the enzyme, reverse transcriptase. The cDNA is then amplified by PCR and the product detected. Agarose gel electrophoresis provides a rapid and simple detection method; however, it is non-quantitative. The assay protocol described in this paper is adapted from that published by Chan et al. Comments on various aspects of the assay are based on experience with the method in our laboratory

  5. Engineering Human Neural Tissue by 3D Bioprinting.

    Science.gov (United States)

    Gu, Qi; Tomaskovic-Crook, Eva; Wallace, Gordon G; Crook, Jeremy M

    2018-01-01

    Bioprinting provides an opportunity to produce three-dimensional (3D) tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a method for fabricating human neural tissue by 3D printing human neural stem cells with a bioink, and subsequent gelation of the bioink for cell encapsulation, support, and differentiation to functional neurons and supporting neuroglia. The bioink uniquely comprises the polysaccharides alginate, water-soluble carboxymethyl-chitosan, and agarose. Importantly, the method could be adapted to fabricate neural and nonneural tissues from other cell types, with the potential to be applied for both research and clinical product development.

  6. Native gel analysis for RISC assembly.

    Science.gov (United States)

    Kawamata, Tomoko; Tomari, Yukihide

    2011-01-01

    Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.

  7. Multichannel microscale system for high throughput preparative separation with comprehensive collection and analysis

    Energy Technology Data Exchange (ETDEWEB)

    Karger, Barry L.; Kotler, Lev; Foret, Frantisek; Minarik, Marek; Kleparnik, Karel

    2003-12-09

    A modular multiple lane or capillary electrophoresis (chromatography) system that permits automated parallel separation and comprehensive collection of all fractions from samples in all lanes or columns, with the option of further on-line automated sample fraction analysis, is disclosed. Preferably, fractions are collected in a multi-well fraction collection unit, or plate (40). The multi-well collection plate (40) is preferably made of a solvent permeable gel, most preferably a hydrophilic, polymeric gel such as agarose or cross-linked polyacrylamide.

  8. Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein.

    Science.gov (United States)

    Lingappa, V R; Lingappa, J R; Prasad, R; Ebner, K E; Blobel, G

    1978-05-01

    mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized alpha-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat alpha-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of alpha-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature alpha-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by alpha-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

  9. Some AFLP amplicons are highly conserved DNA sequences mapping to the same linkage groups in two F2 populations of carrot

    Directory of Open Access Journals (Sweden)

    Santos Carlos A.F.

    2002-01-01

    Full Text Available Amplified fragment length polymorphism (AFLP is a fast and reliable tool to generate a large number of DNA markers. In two unrelated F2 populations of carrot (Daucus carota L., Brasilia x HCM and B493 x QAL (wild carrot, it was hypothesized that DNA 1 digested with the same restriction endonuclease enzymes and amplified with the same primer combination and 2 sharing the same position in polyacrylamide gels should be conserved sequences. To test this hypothesis AFLP fragments from polyacrylamide gels were eluted, reamplified, separated in agarose gels, purified, cloned and sequenced. Among thirty-one paired fragments from each F2 population, twenty-six had identity greater than 91% and five presented identity of 24% to 44%. Among the twenty-six conserved AFLPs only one mapped to different linkage groups in the two populations while four of the five less-conserved bands mapped to different linkage groups. Of eight SCAR (sequence characterized amplified regions primers tested, one conserved AFLP resulted in co-dominant markers in both populations. Screening among 14 carrot inbreds or cultivars with three AFLP-SCAR primers revealed clear and polymorphic PCR products, with similar molecular sizes on agarose gels. The development of co-dominant markers based on conserved AFLP fragments will be useful to detect seed mixtures among hybrids, to improve and to merge linkage maps and to study diversity and phylogenetic relationships.

  10. 3D printing facilitated scaffold-free tissue unit fabrication

    International Nuclear Information System (INIS)

    Tan, Yu; Richards, Dylan J; Mei, Ying; Trusk, Thomas C; Visconti, Richard P; Yost, Michael J; Drake, Christopher J; Argraves, William Scott; Markwald, Roger R; Kindy, Mark S

    2014-01-01

    Tissue spheroids hold great potential in tissue engineering as building blocks to assemble into functional tissues. To date, agarose molds have been extensively used to facilitate fusion process of tissue spheroids. As a molding material, agarose typically requires low temperature plates for gelation and/or heated dispenser units. Here, we proposed and developed an alginate-based, direct 3D mold-printing technology: 3D printing microdroplets of alginate solution into biocompatible, bio-inert alginate hydrogel molds for the fabrication of scaffold-free tissue engineering constructs. Specifically, we developed a 3D printing technology to deposit microdroplets of alginate solution on calcium containing substrates in a layer-by-layer fashion to prepare ring-shaped 3D hydrogel molds. Tissue spheroids composed of 50% endothelial cells and 50% smooth muscle cells were robotically placed into the 3D printed alginate molds using a 3D printer, and were found to rapidly fuse into toroid-shaped tissue units. Histological and immunofluorescence analysis indicated that the cells secreted collagen type I playing a critical role in promoting cell–cell adhesion, tissue formation and maturation. (paper)

  11. Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

    Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

  12. Bacterial community dynamics during polysaccharide degradation at contrasting sites in the Southern and Atlantic Oceans.

    Science.gov (United States)

    Wietz, Matthias; Wemheuer, Bernd; Simon, Heike; Giebel, Helge-Ansgar; Seibt, Maren A; Daniel, Rolf; Brinkhoff, Thorsten; Simon, Meinhard

    2015-10-01

    The bacterial degradation of polysaccharides is central to marine carbon cycling, but little is known about the bacterial taxa that degrade specific marine polysaccharides. Here, bacterial growth and community dynamics were studied during the degradation of the polysaccharides chitin, alginate and agarose in microcosm experiments at four contrasting locations in the Southern and Atlantic Oceans. At the Southern polar front, chitin-supplemented microcosms were characterized by higher fractions of actively growing cells and a community shift from Alphaproteobacteria to Gammaproteobacteria and Bacteroidetes. At the Antarctic ice shelf, chitin degradation was associated with growth of Bacteroidetes, with 24% higher cell numbers compared with the control. At the Patagonian continental shelf, alginate and agarose degradation covaried with growth of different Alteromonadaceae populations, each with specific temporal growth patterns. At the Mauritanian upwelling, only the alginate hydrolysis product guluronate was consumed, coincident with increasing abundances of Alteromonadaceae and possibly cross-feeding SAR11. 16S rRNA gene amplicon libraries indicated that growth of the Bacteroidetes-affiliated genus Reichenbachiella was stimulated by chitin at all cold and temperate water stations, suggesting comparable ecological roles over wide geographical scales. Overall, the predominance of location-specific patterns showed that bacterial communities from contrasting oceanic biomes have members with different potentials to hydrolyse polysaccharides. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  13. Characterizing Thermal Augmentation of Convection-Enhanced Drug Delivery with the Fiberoptic Microneedle Device

    Directory of Open Access Journals (Sweden)

    R. Lyle Hood

    2015-09-01

    Full Text Available Convection-enhanced delivery (CED is a promising technique leveraging pressure-driven flow to increase penetration of infused drugs into interstitial spaces. We have developed a fiberoptic microneedle device for inducing local sub-lethal hyperthermia to further improve CED drug distribution volumes, and this study seeks to quantitatively characterize this approach in agarose tissue phantoms. Infusions of dye were conducted in 0.6% (w/w agarose tissue phantoms with isothermal conditions at 15 °C, 20 °C, 25 °C, and 30 °C. Infusion metrics were quantified using a custom shadowgraphy setup and image-processing algorithm. These data were used to build an empirical predictive temporal model of distribution volume as a function of phantom temperature. A second set of proof-of-concept experiments was conducted to evaluate a novel fiberoptic device capable of generating local photothermal heating during fluid infusion. The isothermal infusions showed a positive correlation between temperature and distribution volume, with the volume at 30 °C showing a 7-fold increase at 100 min over the 15 °C isothermal case. Infusions during photothermal heating (1064 nm at 500 mW showed a similar effect with a 3.5-fold increase at 4 h over the control (0 mW. These results and analyses serve to provide insight into and characterization of heat-mediated enhancement of volumetric dispersal.

  14. Infrared spectral imaging as a novel approach for histopathological recognition in colon cancer diagnosis

    Science.gov (United States)

    Nallala, Jayakrupakar; Gobinet, Cyril; Diebold, Marie-Danièle; Untereiner, Valérie; Bouché, Olivier; Manfait, Michel; Sockalingum, Ganesh Dhruvananda; Piot, Olivier

    2012-11-01

    Innovative diagnostic methods are the need of the hour that could complement conventional histopathology for cancer diagnosis. In this perspective, we propose a new concept based on spectral histopathology, using IR spectral micro-imaging, directly applied to paraffinized colon tissue array stabilized in an agarose matrix without any chemical pre-treatment. In order to correct spectral interferences from paraffin and agarose, a mathematical procedure is implemented. The corrected spectral images are then processed by a multivariate clustering method to automatically recover, on the basis of their intrinsic molecular composition, the main histological classes of the normal and the tumoral colon tissue. The spectral signatures from different histological classes of the colonic tissues are analyzed using statistical methods (Kruskal-Wallis test and principal component analysis) to identify the most discriminant IR features. These features allow characterizing some of the biomolecular alterations associated with malignancy. Thus, via a single analysis, in a label-free and nondestructive manner, main changes associated with nucleotide, carbohydrates, and collagen features can be identified simultaneously between the compared normal and the cancerous tissues. The present study demonstrates the potential of IR spectral imaging as a complementary modern tool, to conventional histopathology, for an objective cancer diagnosis directly from paraffin-embedded tissue arrays.

  15. Polymerase Chain Reaction (Pcr) Assay to Detect Hepatitis C Virus

    International Nuclear Information System (INIS)

    Lina MR; Dadang S; Budiman Bela

    2004-01-01

    Research on the detection of hepatitis C virus in blood serum using PCR technique has been carried out. Amount of 50 blood serum from laboratory of Indonesia Red Cross (Palang Merah Indonesia = PMI) and RSCM hospital as samples, were used in this research. Lysis of virus cell and extraction of RNA virus as a preliminary treatment of the sample, was done with BOOM method using guanidine thiocyanate and diatomaceous earth, respectively. Synthesis of cDNA from RNA as an extraction product mentioned above, was carried out by means of reverse-transcriptase and RNA-se inhibitor. Amplification of cDNA was done with nested PCR technique that was performed with two times PCR processes using two pairs of oligonucleotide primers for each process. The amplified DNA was detected by agarose gel electrophoresis and ethidium bromide staining. Subsequently, the DNA was visualized with UV transilluminator. Result shows that of 50 blood serum samples, 13 serum were positive for RNA HCV that were performed with the present of specific DNA band on agarose gel. (author)

  16. Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography.

    Directory of Open Access Journals (Sweden)

    Indhu Kanakaraj

    Full Text Available Immobilized Metal Affinity Chromatography (IMAC has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and "histidine tags" genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu(2+-iminodiacetic acid (IDA agarose spin column, 94-99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu(2+-IDA agarose can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.

  17. Capillary crystallization and molecular-replacement solution of haemoglobin II from the clam Lucina pectinata

    International Nuclear Information System (INIS)

    Gavira, José A.; Jesus, Walleska de; Camara-Artigas, Ana; López-Garriga, Juan; García-Ruiz, Juan M.

    2006-01-01

    The haemoglobin II from the clam L. pectinata has been crystallized using counter-diffusion in single capillary in the presence of agarose to improve crystal quality. Initial phases have been obtained by molecular replacement. Haemoglobin II is one of three haemoglobins present in the cytoplasm of the Lucina pectinata mollusc that inhabits the Caribbean coast. Using HBII purified from its natural source, crystallization screening was performed using the counter-diffusion method with capillaries of 0.2 mm inner diameter. Crystals of HbII suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to improve their quality. The crystals belong to the tetragonal space group P4 2 2 1 2, with unit-cell parameters a = b = 73.92, c = 152.35 Å, and diffracted X-rays to a resolution of better than 2.0 Å. The asymmetric unit is a homodimer with a corresponding Matthews coefficient (V M ) of 3.15 Å 3 Da −1 and a solvent content of 61% by volume

  18. Self-assembly of tissue spheroids on polymeric membranes.

    Science.gov (United States)

    Messina, Antonietta; Morelli, Sabrina; Forgacs, Gabor; Barbieri, Giuseppe; Drioli, Enrico; De Bartolo, Loredana

    2017-07-01

    In this study, multicellular tissue spheroids were fabricated on polymeric membranes in order to accelerate the fusion process and tissue formation. To this purpose, tissue spheroids composed of three different cell types, myoblasts, fibroblasts and neural cells, were formed and cultured on agarose and membranes of polycaprolactone (PCL) and chitosan (CHT). Membranes prepared by a phase-inversion technique display different physicochemical, mechanical and transport properties, which can affect the fusion process. The membranes accelerated the fusion process of a pair of spheroids with respect to the inert substrate. In this process, a critical role is played by the membrane properties, especially by their mechanical characteristics and oxygen and carbon dioxide mass transfer. The rate of fusion was quantified and found to be similar for fibroblast, myoblast and neural tissue spheroids on membranes, which completed the fusion within 3 days. These spheroids underwent faster fusion and maturation on PCL membrane than on agarose, the rate of fusion being proportional to the value of oxygen and carbon dioxide permeances and elastic characteristics. Consequently, tissue spheroids on the membranes expressed high biological activity in terms of oxygen uptake, making them more suitable as building blocks in the fabrication of tissues and organs. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  19. A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

    Directory of Open Access Journals (Sweden)

    R.J. Burgdorf

    2014-09-01

    Full Text Available Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA and non-metric multidimensional scaling (NMDS were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM. Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05 from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05. The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

  20. A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies.

    Science.gov (United States)

    Burgdorf, R J; Laing, M D; Morris, C D; Jamal-Ally, S F

    2014-01-01

    Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

  1. Rotational Diffusion of Macromolecules and Nanoparticles Modeled as Non-Overlapping Bead Arrays in an Effective Medium

    Directory of Open Access Journals (Sweden)

    Umar Twahir

    2011-05-01

    Full Text Available In this work, the retarding influence of a gel on the rotational motion of a macromolecule is investigated within the framework of the Effective Medium (EM model. This is an extension of an earlier study that considered the effect of a gel on the translational motion of a macromolecule [Allison, S. et al. J. Phys. Chem. B 2008, 112, 5858-5866]. The macromolecule is modeled as an array of non-overlapping spherical beads with no restriction placed on their size or configuration. Specific applications include the rotational motion of right circular cylinders and wormlike chains modeled as strings of identical touching beads. The procedure is then used to examine the electric birefringence decay of a 622 base pair DNA fragment in an agarose gel. At low gel concentration (M £ 0.010 gm/mL, good agreement between theory and experiment is achieved if the persistence length of DNA is taken to be 65 nm and the gel fiber radius of agarose is taken to be 2.5 nm. At higher gel concentrations, the EM model substantially underestimates the rotational relaxation time of DNA and this can be attributed to the onset of direct interactions that become significant when the effective particle size becomes comparable to the mean gel fiber spacing.

  2. Microfluidic liquid-air dual-gradient chip for synergic effect bio-evaluation of air pollutant.

    Science.gov (United States)

    Liu, Xian-Jun; Hu, Shan-Wen; Xu, Bi-Yi; Zhao, Ge; Li, Xiang; Xie, Fu-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2018-05-15

    In this paper, a novel prototype liquid-air dual gradient chip is introduced, which has paved the way for effective synergic effect bio-evaluation of air pollutant. The chip is composed of an array of the agarose liquid-air interfaces, top air gradient layer and bottom liquid gradient layer. The novel agarose liquid-air interface allows for non-biased exposure of cells to all the substances in the air and diffusive interactions with the liquid phase; while the dual liquid-air gradient provides powerful screening abilities, which well reduced errors, saved time and cost from repeated experiment. Coupling the two functions, the chip subsequently facilitates synergic effect evaluation of both liquid and air factors on cells. Here cigarette smoke was taken as the model air pollutant, and its strong synergic effects with inflammatory level of A549 lung cancer cells on their fate were successfully quantified for the first time. These results well testified that the proposed dual-gradient chip is powerful and indispensable for bio-evaluation of air pollutant. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Semester-long inquiry-based molecular biology laboratory: Transcriptional regulation in yeast.

    Science.gov (United States)

    Oelkers, Peter M

    2017-03-04

    A single semester molecular biology laboratory has been developed in which students design and execute a project examining transcriptional regulation in Saccharomyces cerevisiae. Three weeks of planning are allocated to developing a hypothesis through literature searches and use of bioinformatics. Common experimental plans address a cell process and how three genes that encode for proteins involved in that process are transcriptionally regulated in response to changing environmental conditions. Planning includes designing oligonucleotides to amplify the putative promoters of the three genes of interest. After the PCR, each product is cloned proximal to β-galactosidase in a yeast reporter plasmid. Techniques used include agarose electrophoresis, extraction of DNA from agarose, plasmid purification from bacteria, restriction digestion, ligation, and bacterial transformation. This promoter/reporter plasmid is then transformed into yeast. Transformed yeast are cultured in conditions prescribed in the experimental design, lysed and β-galactosidase activity is measured. The course provides an independent research experience in a group setting. Notebooks are maintained on-line with regular feedback. Projects culminate with the presentation of a poster worth 60% of the grade. Over the last three years, about 65% of students met expectations for experimental design, data acquisition, and analysis. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):145-151, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

  4. Dynamics of a camphoric acid boat at the air-water interface

    Science.gov (United States)

    Akella, V. S.; Singh, Dhiraj K.; Mandre, Shreyas; Bandi, M. M.

    2018-05-01

    We report experiments on an agarose gel tablet loaded with camphoric acid (c-boat) spontaneously set into motion by surface tension gradients on the water surface. We observe three distinct modes of c-boat motion: harmonic mode where the c-boat speed oscillates sinusoidally in time, a steady mode where the c-boat maintains constant speed, and an intermittent mode where the c-boat maintains near-zero speed between sudden jumps in speed. Whereas all three modes have been separately reported before in different systems, controlled release of Camphoric Acid (CA) from the agarose gel matrix allowed the observation of all the three modes in the same system. These three modes are a result of a competition between the driving (surface tension gradients) and drag forces acting on the c-boat. Moreover we suggest that there exist two time scales corresponding to spreading of CA and boat motion and the mismatch of these two time scales give rise to the three modes in boat motion. We reproduced all the modes of motion by varying the air-water interfacial tension using Sodium Dodecyl Sulfate (SDS).

  5. Comparison of Superparamagnetic Iron Oxide Labeling Efficiency between Poly-L-Lysine and Protamine Sulfate for Human Mesenchymal Stem Cells: Quantitative Analysis Using Multi-Echo T2 Magnetic Resonance Imaging

    International Nuclear Information System (INIS)

    Suh, Ji Yeon; Lee, Jeong Hyun; Lee, Chang Kyung; Shin, Ji Hoon; Choi, Choong Gon; Kim, Jeong Kon

    2013-01-01

    To quantify in vitro labeling efficiency of protamine sulfate (PS) and poly-L-lysine (PLL) for labeling of human mesenchymal stem cells (hMSCs) with superparamagnetic iron oxide (SPIO) using multi-echo T2 magnetic resonance (MR) imaging at 4.7 T. The hMSCs were incubated with SPIO-PS or SPIO-PLL complexes. Their effects on the cell metabolism and differentiation capability were evaluated, respectively. The decrease of iron concentrations in the labeled cells were assessed immediately, and at 4 d after labeling using multi-echo T2 MR imaging at 4.7 T. The results were compared with those of Prussian blue colorimetry. The hMSCs were labeled more efficiently by SPIO-PLL than SPIO-PS without any significant effect on cell metabolism and differentiation capabilities. It was feasible to quantify the iron concentrations in SPIO-agarose-phantoms and in agarose mixture with the labeled cells from T2 maps obtained from multi-echo T2 MRI. However, the iron concentration of the labeled cells was significantly higher by T2-maps than the results of Prussian blue colorimetry. The hMSCs can be effectively labeled with SPIO-PLL complexes more than with SPIO-PS without significant change in cell metabolism and differentiation. In vitro quantification of the iron concentrations of the labeled is feasible from multi-echo T2 MRI, but needs further investigation.

  6. Comparison of Superparamagnetic Iron Oxide Labeling Efficiency between Poly-L-Lysine and Protamine Sulfate for Human Mesenchymal Stem Cells: Quantitative Analysis Using Multi-Echo T2 Magnetic Resonance Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Ji Yeon; Lee, Jeong Hyun; Lee, Chang Kyung; Shin, Ji Hoon; Choi, Choong Gon; Kim, Jeong Kon [Dept. of Radiology and Research Institute of Radiology, University of Ulsan College of Medicine, Asan Medical Center, Seoul (Korea, Republic of)

    2013-02-15

    To quantify in vitro labeling efficiency of protamine sulfate (PS) and poly-L-lysine (PLL) for labeling of human mesenchymal stem cells (hMSCs) with superparamagnetic iron oxide (SPIO) using multi-echo T2 magnetic resonance (MR) imaging at 4.7 T. The hMSCs were incubated with SPIO-PS or SPIO-PLL complexes. Their effects on the cell metabolism and differentiation capability were evaluated, respectively. The decrease of iron concentrations in the labeled cells were assessed immediately, and at 4 d after labeling using multi-echo T2 MR imaging at 4.7 T. The results were compared with those of Prussian blue colorimetry. The hMSCs were labeled more efficiently by SPIO-PLL than SPIO-PS without any significant effect on cell metabolism and differentiation capabilities. It was feasible to quantify the iron concentrations in SPIO-agarose-phantoms and in agarose mixture with the labeled cells from T2 maps obtained from multi-echo T2 MRI. However, the iron concentration of the labeled cells was significantly higher by T2-maps than the results of Prussian blue colorimetry. The hMSCs can be effectively labeled with SPIO-PLL complexes more than with SPIO-PS without significant change in cell metabolism and differentiation. In vitro quantification of the iron concentrations of the labeled is feasible from multi-echo T2 MRI, but needs further investigation.

  7. Study on apoptosis of prostate cancer cell induced by 125I seed irradiation

    International Nuclear Information System (INIS)

    Liao Anyan; Wang Junjie; Wang Jidong; Zhuang Hongqing; Zhao Yong

    2007-01-01

    Objective: To explore the mechanism of apoptosis induced by 125 I seed irradiation on PC3 cells. Methods: Human prostate cancer cell line PC3 was treated by irradiation of 125 I (2.77 cGy/h) with various dose. Agarose gel electrophoresis of DNA and flows cytometry were used to detect the apoptosis of PC3 cells and indirect immunofluorescence assay was used to detect the expression of Bcl-2. The activity of Caspase-3 was measured by Caspase Colorimetric Assay Kits. Results: Apoptosis of PC3 cells could be efficiently induced by 125 I seed irradiation. The apoptotic peaks were found by flow cytometry and DNA ladder appeared on 1.8% agarose gel. The activity of Caspase-3 on PC3 cells treated by 125 I seed irradiation was not changed significantly. Bcl-2 gene expression was down-regulated with the sample concentration increased. Conclusion: 125 I irradiation can induce the apoptosis of PC3 cells and the mechanism of apoptosis is related with down regulation of Bcl-2 gene expression and is not related with Caspase-3 activity. (authors)

  8. Apoptosis of nasopharyngeal carcinoma cell line (CNE-2) induced by neutron irradiation

    International Nuclear Information System (INIS)

    Liang Ke; He Shaoqin; Feng Yan; Tang Jinhua; Feng Qinfu; Shen Yu; Yin Weibo; Xu Guozhen; Liu Xinfan; Wang Luhua; Gao Li

    1999-01-01

    Objective: To study the apoptotic response of the nasopharyngeal carcinoma cell line (CNE-2) induced by neutron irradiation. Methods: CNE-2 cells were cultured as usual. Using the techniques of DNA agarose gel electrophoresis and DNA special fluorescent staining, the status of apoptosis in CNE-2 cells after neutron irradiation was detected. Results: It was shown that the apoptosis can be induced in CNE-2 cell after neutron radiation. Six hrs, after different doses of neutron (0/0.667/1.333/2.000/2.667/3.333 Gy) and X-ray 0/2/4/6/8/10 Gy) irradiation the apoptotic rates were 2.4%, 6.3%, 7.1%, 9.5%, 13.5%, 14.6% and 2.4%, 3.8%, 5.7%, 7.8%, 10.4%, 11.7%, respectively; at 48 hrs they were 18.3%, 21.5%, 22.8%, 29.3%, 34.2% and 13.7%, 17.6%, 21.3%, 25.6%, 28.9%, respectively. At 10 hrs after neutron irradiation the DNA ladder of apoptosis could be detected between 0.667-3.333 Gy doses in CNE-2 cells by DNA agarose gel electrophoresis. Conclusion: Neutron radiation can induce apoptosis in tumor cells. Compared with the X-ray, neutron induces apoptosis in larger extent than X-ray in the same condition; meanwhile, apoptosis after irradiation is dose and time dependent

  9. 3D-printed microwell arrays for Ciona microinjection and timelapse imaging.

    Directory of Open Access Journals (Sweden)

    Clint Gregory

    Full Text Available Ascidians such as Ciona are close chordate relatives of the vertebrates with small, simple embryonic body plans and small, simple genomes. The tractable size of the embryo offers considerable advantages for in toto imaging and quantitative analysis of morphogenesis. For functional studies, Ciona eggs are considerably more challenging to microinject than the much larger eggs of other model organisms such as zebrafish and Xenopus. One of the key difficulties is in restraining the eggs so that the microinjection needle can be easily introduced and withdrawn. Here we develop and test a device to cast wells in agarose that are each sized to hold a single egg. This injection mold is fabricated by micro-resolution stereolithography with a grid of egg-sized posts that cast corresponding wells in agarose. This 3D printing technology allows the rapid and inexpensive testing of iteratively refined prototypes. In addition to their utility in microinjection, these grids of embryo-sized wells are also valuable for timelapse imaging of multiple embryos.

  10. In vitro simulation of distribution processes following intramuscular injection

    Directory of Open Access Journals (Sweden)

    Probst Mareike

    2016-09-01

    Full Text Available There is an urgent need for in vitro dissolution test setups for intramuscularly applied dosage forms. Especially biorelevant methods are needed to predict the in vivo behavior of newly developed dosage forms in a realistic way. There is a lack of knowledge regarding critical in vivo parameters influencing the release and absorption behavior of an intramuscularly applied drug. In the presented work the focus was set on the simulation of blood perfusion and muscle tissue. A solid agarose gel, being incorporated in an open-pored foam, was used to mimic the gel phase of muscle tissue and implemented in a flow through cell. An aqueous solution of fluorescein sodium was injected. Compared to recently obtained in vivo results the distribution of the model substance was very slow. Furthermore an agarose gel of lower viscosity an open-pored foam and phosphate buffer saline pH 7.4 were implemented in a multi-channel-ceramic membrane serving as a holder for the muscle imitating material. Blood simulating release medium was perfused through the ceramic membrane including filling materials. Transport of the dissolved fluorescein sodium was, in case of the gel, not only determined by diffusion but also by convective transport processes. The more realistic the muscle simulating materials were constituted the less reproducible results were obtained with the designed test setups.

  11. Characterization of a novel alkaline arylsulfatase from Marinomonas sp. FW-1 and its application in the desulfation of red seaweed agar.

    Science.gov (United States)

    Wang, Xueyan; Duan, Delin; Xu, Jiachao; Gao, Xin; Fu, Xiaoting

    2015-10-01

    A bacterial strain capable of hydrolyzing sulfate ester bonds of p-nitrophenyl sulfate (pNPS) and agar was isolated from the coast area of Qingdao, China. It was identified as Marinomonas based on its 16S rRNA gene sequence and named as Marinomonas sp. FW-1. An arylsulfatase with a recovery of 13 % and a fold of 12 was purified to a homogeneity using ion exchange and gel filtration chromatographies. The enzyme was composed of a single polypeptide chain with the molecular mass of 33 kDa estimated using SDS-PAGE. The optimal pH and temperature of arylsulfatase were pH 9.0 and 45, respectively. Arylsulfatase was stable over pH 8-11 and at temperature below 55 °C. The K m and V max of this enzyme for the hydrolysis of pNPS were determined to be 13.73 and 270.27 μM/min, respectively. The desulfation ratio against agar from red seaweed Gelidium amansii and Gracilaria lemaneiformis were 86.11 and 89.61 %, respectively. There was no difference between the DNA electrophoresis spectrum on the gel of the arylsulfatase-treated G. amansii agar and that of the commercial agarose. Therefore, this novel alkaline arylsulfatase might have a great potential for application in enzymatic conversion of agar to agarose.

  12. The effect of different methods and image analyzers on the results of the in vivo comet assay.

    Science.gov (United States)

    Kyoya, Takahiro; Iwamoto, Rika; Shimanura, Yuko; Terada, Megumi; Masuda, Shuichi

    2018-01-01

    The in vivo comet assay is a widely used genotoxicity test that can detect DNA damage in a range of organs. It is included in the Organisation for Economic Co-operation and Development Guidelines for the Testing of Chemicals. However, various protocols are still used for this assay, and several different image analyzers are used routinely to evaluate the results. Here, we verified a protocol that largely contributes to the equivalence of results, and we assessed the effect on the results when slides made from the same sample were analyzed using two different image analyzers (Comet Assay IV vs Comet Analyzer). Standardizing the agarose concentrations and DNA unwinding and electrophoresis times had a large impact on the equivalence of the results between the different methods used for the in vivo comet assay. In addition, there was some variation in the sensitivity of the two different image analyzers tested; however this variation was considered to be minor and became negligible when the test conditions were standardized between the two different methods. By standardizing the concentrations of low melting agarose and DNA unwinding and electrophoresis times between both methods used in the current study, the sensitivity to detect the genotoxicity of a positive control substance in the in vivo comet assay became generally comparable, independently of the image analyzer used. However, there may still be the possibility that other conditions, except for the three described here, could affect the reproducibility of the in vivo comet assay.

  13. Influence of experimental conditions on data variability in the liver comet assay.

    Science.gov (United States)

    Guérard, M; Marchand, C; Plappert-Helbig, U

    2014-03-01

    The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters. Copyright © 2013 Wiley Periodicals, Inc.

  14. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle

    International Nuclear Information System (INIS)

    Chadwick, C.C.; Saito, A.; Fleischer, S.

    1990-01-01

    The release of Ca 2+ from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca 2+ release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP 3 ) is believed to mobilize Ca 2+ from internal stores and thereby modulate contraction. The authors describe the isolation of an IP 3 receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and the IP 3 receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The receptor is an oligomer of a single polypeptide with a M r of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of ∼250 x 250 angstrom with fourfold symmetry. The IP 3 receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding

  15. Studying biomolecule localization by engineering bacterial cell wall curvature.

    Directory of Open Access Journals (Sweden)

    Lars D Renner

    Full Text Available In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.

  16. Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

    Science.gov (United States)

    Amarger, V; Mercier, L

    1995-01-01

    We have applied the recently developed technique of random amplified polymorphic DNA (RAPD) for the discrimination between two jojoba clones at the genomic level. Among a set of 30 primers tested, a simple reproducible pattern with three distinct fragments for clone D and two distinct fragments for clone E was obtained with primer OPB08. Since RAPD products are the results of arbitrarily priming events and because a given primer can amplify a number of non-homologous sequences, we wondered whether or not RAPD bands, even those of similar size, were derived from different loci in the two clones. To answer this question, two complementary approaches were used: i) cloning and sequencing of the amplification products from clone E; and ii) complementary Southern analysis of RAPD gels using cloned or amplified fragments (directly recovered from agarose gels) as RFLP probes. The data reported here show that the RAPD reaction generates multiple amplified fragments. Some fragments, although resolved as a single band on agarose gels, contain different DNA species of the same size. Furthermore, it appears that the cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals.

  17. Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

    Science.gov (United States)

    Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

    2012-08-01

    Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

  18. Preparation of Fe3O4/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride) by emulsifier-free emulsion polymerization and its interaction with DNA

    International Nuclear Information System (INIS)

    Li Xiaolong; Liu Guoqiang; Yan Wei; Chu, Paul K.; Yeung, Kelvin W.K.; Wu Shuilin; Yi Changfeng; Xu Zushun

    2012-01-01

    Cationic magnetic polymer particles Fe 3 O 4 /poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride), a type of potential gene carrier, were prepared by emulsifier-free emulsion polymerization with oleic acid modified magnetite Fe 3 O 4 , styrene, butyl acrylate and [2-(methacryloxy)ethyl]trimethylammonium chloride) (METAC). The morphology of the particles was characterized by transmission electron microscopy and the composites of particles were characterized by FT-IR spectroscopy, X-ray diffraction. These results showed that magnetic particles were well dispersed in polymers with the content of about 15%(wt/wt). The composites exhibited superparamagnetism and possessed a certain level of magnetic response. The interactions between the particles with calf-thymus DNA (ct DNA) were confirmed by zeta potential measurement, UV–vis spectroscopy and fluorescence spectroscopy. The DNA-binding capacity determined by the agarose gel electrophoresis showed good binding capacity of the emulsion to DNA. These results suggested the potential of the cationic magnetic polymer emulsion as gene target delivery carrier. - Highlights: ► A new type of cationic magnetic polymer particles was synthesized by emulsifier-free emulsion polymerization. ► Structural, morphological, and magnetic properties of the composite were evaluated. ► The interaction between cationic magnetic polymer particles with DNA was confirmed by zeta potential measurements. ► UV–vis spectrophotometry, fluorescent spectroscopy and agarose gel electrophoresis. ► This process may have potential applications to gene carrier and DNA separation.

  19. OBTENÇÃO DE PLANTAS DE LIMÃO CRAVO (Citrus limonia Osbeck E TANGERINA CLEÓPATRA (Citrus reshni Hort. A PARTIR DO CULTIVO DE PROTOPLASTOS DE SUSPENSÃO CELULAR PLANT REGENERATION OF 'RANGPUR' LIME (Citrus limonia Osbeck AND 'CLEÓPATRA' MANDARIN (Citrus reshni Hort. THROUGH PROTOPLASTS OF CELL SUSPENSION

    Directory of Open Access Journals (Sweden)

    Rodrigo Rocha Latado

    1999-01-01

    Full Text Available Este trabalho descreve uma metodologia para a regeneração de plantas de tangerina 'Cleópatra' e limão 'Cravo', a partir do cultivo de protoplastos de suspensão celular. Para tal, calos nucelares foram induzidos em meio contendo BAP e cultivados em meio sem reguladores de crescimento. Protoplastos foram isolados de suspensões celulares e cultivados em gotas de agarose, com densidade de 2 X 105 protoplastos.ml-1. O meio MT, contendo ácido giberélico e água de coco, foi eficiente na germinação de embriões somáticos. Os métodos de aclimatação de plantas testados apresentaram baixa eficiência. Como resultado final, 17 plantas adaptadas de tangerina e 8 de limão foram obtidas.The present research describes the regeneration of 'Cleópatra' mandarin and 'Rangpur' lime plants from cell suspension protoplasts. Nucelar calli were induced on a medium containing BAP and maintained on growth regulator free medium. Protoplasts were isolated from embryogenic suspension and plated at a concentration of 2 X 105 protoplasts.ml-1, on agarose droplets. The MT medium with gibberellic acid and coconut water was efficient to stimulate somatic embryo conversion. Rooted plants acclimation had low efficiency. Seventeen mandarin plants and eight lime plants were obtained.

  20. Growth of magnetite films by a hydrogel method

    Energy Technology Data Exchange (ETDEWEB)

    Velásquez, A.A., E-mail: avelas26@eafit.edu.edu.co [Grupo de Electromagnetismo Aplicado, Universidad EAFIT, A.A. 3300, Medellín (Colombia); Marín, C.C. [Grupo de Electromagnetismo Aplicado, Universidad EAFIT, A.A. 3300, Medellín (Colombia); Urquijo, J.P. [Grupo de Estado Sólido, Instituto de Física, Universidad de Antioquia, A.A. 1226, Medellín (Colombia)

    2017-06-15

    Magnetite (Fe{sub 3}O{sub 4}) films were grown on glass substrates by formation and condensation of complex of iron oxides in an agarose hydrogel. The obtained films were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Room Temperature Mössbauer Spectroscopy (TMS), Vibrating Sample Magnetometry (VSM), Atomic Force Microscopy (AFM) and Voltage vs. Current measurements by the four-point method. FTIR and TGA measurements showed that some polymer chains of agarose remain linked to the surface of the magnetic particles of the films after heat treatment. SEM measurements showed that the films are composed by quasi spherical particles with sizes around 55 nm. Mössbauer spectroscopy measurements showed two sextets with broaden lines, which were assigned to magnetite with a distributed particle size, and two doublets, which were assigned to superparamagnetic phases of magnetite. For the specific dimensions of the films prepared, measurements of Voltage vs. Current showed an ohmic behavior for currents between 0 and 200 nA, with a resistance of 355 kΩ.

  1. Development of a bi-functional silica monolith for electro-osmotic pumping and DNA clean-up/extraction using gel-supported reagents in a microfluidic device.

    Science.gov (United States)

    Oakley, Jennifer A; Shaw, Kirsty J; Docker, Peter T; Dyer, Charlotte E; Greenman, John; Greenway, Gillian M; Haswell, Stephen J

    2009-06-07

    A silica monolith used to support both electro-osmotic pumping (EOP) and the extraction/elution of DNA coupled with gel-supported reagents is described. The benefits of the combined EOP extraction/elution system were illustrated by combining DNA extraction and gene amplification using the polymerase chain reaction (PCR) process. All the reagents necessary for both processes were supported within pre-loaded gels that allow the reagents to be stored at 4 degrees C for up to four weeks in the microfluidic device. When carrying out an analysis the crude sample only needed to be hydrodynamically introduced into the device which was connected to an external computer controlled power supply via platinum wire electrodes. DNA was extracted with 65% efficiency after loading lysed cells onto a silica monolith. Ethanol contained within an agarose gel matrix was then used to wash unwanted debris away from the sample by EOP (100 V cm(-1) for 5 min). The retained DNA was subsequently eluted from the monolith by water contained in a second agarose gel, again by EOP using an electric field of 100 V cm(-1) for 5 min, and transferred into the PCR reagent containing gel. The eluted DNA in solution was successfully amplified by PCR, confirming that the concept of a complete self-contained microfluidic device could be realised for DNA sample clean up and amplification, using a simple pumping and on-chip reagent storage methodology.

  2. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Directory of Open Access Journals (Sweden)

    Hoseok Choi

    2016-04-01

    Full Text Available Assaying the glycogen synthase kinase-3 (GSK3 activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.

  3. Bioprinting Organotypic Hydrogels with Improved Mesenchymal Stem Cell Remodeling and Mineralization Properties for Bone Tissue Engineering.

    Science.gov (United States)

    Duarte Campos, Daniela Filipa; Blaeser, Andreas; Buellesbach, Kate; Sen, Kshama Shree; Xun, Weiwei; Tillmann, Walter; Fischer, Horst

    2016-06-01

    3D-manufactured hydrogels with precise contours and biological adhesion motifs are interesting candidates in the regenerative medicine field for the culture and differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). 3D-bioprinting is a powerful technique to approach one step closer the native organization of cells. This study investigates the effect of the incorporation of collagen type I in 3D-bioprinted polysaccharide-based hydrogels to the modulation of cell morphology, osteogenic remodeling potential, and mineralization. By combining thermo-responsive agarose hydrogels with collagen type I, the mechanical stiffness and printing contours of printed constructs can be improved compared to pure collagen hydrogels which are typically used as standard materials for MSC osteogenic differentiation. The results presented here show that MSC not only survive the 3D-bioprinting process but also maintain the mesenchymal phenotype, as proved by live/dead staining and immunocytochemistry (vimentin positive, CD34 negative). Increased solids concentrations of collagen in the hydrogel blend induce changes in cell morphology, namely, by enhancing cell spreading, that ultimately contribute to enhanced and directed MSC osteogenic differentiation. 3D-bioprinted agarose-collagen hydrogels with high-collagen ratio are therefore feasible for MSC osteogenic differentiation, contrarily to low-collagen blends, as proved by two-photon microscopy, Alizarin Red staining, and real-time polymerase chain reaction. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Real-time UV imaging of piroxicam diffusion and distribution from oil solutions into gels mimicking the subcutaneous matrix.

    Science.gov (United States)

    Ye, Fengbin; Larsen, Susan Weng; Yaghmur, Anan; Jensen, Henrik; Larsen, Claus; Østergaard, Jesper

    2012-05-12

    A novel real-time UV imaging approach for non-intrusive investigation of the diffusion and partitioning phenomena occurring during piroxicam release from medium chain triglyceride (MCT) solution into two hydrogel matrices is described. Two binary polymer/buffer gel matrices, 0.5% (w/v) agarose and 25% (w/v) Pluronic F127, were applied as simple models mimicking the subcutaneous tissue. The evolution of the absorbance maps as a function of time provided detailed information on the piroxicam release processes upon the exposure of the gel matrices to MCT. Using calibration curves, the concentration maps of piroxicam in the UV imaging area were determined. Regression of the longitudinal concentration-distance profiles, which were obtained using expressions derived from Fick's second law, provided the diffusivity and the distribution coefficients of piroxicam penetrated into the gels. The obtained MCT-agarose (pH 7.4) distribution coefficient of 1.4 was identical to the MCT-aqueous (pH 7.4) distribution coefficient determined by the shake-flask method whereas that of the MCT-Pluronic F127 system was four times less. The experimental data show that UV imaging may have considerable potential for investigating the transport properties of drug formulations intended for the subcutaneous administration. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Effect of immobilized lipase supplementation of diets on the performance of the Japanese quails

    International Nuclear Information System (INIS)

    Abu-Taleb, A.M.; Ezzat, I.E.; Saleh, M.

    2004-01-01

    In the present study, lipase was immobilized onto two different supports, agarose and gelatin. Some physico-chemical properties of the free and immobilized lipase such as optimum temperature, optimum ph and storage stability were studied. Storage of the enzymes for 2 months showed that the free enzyme lost its activity, while the immobilized on the gelatin showed better resistance towards ph and temperature variations than that immobilized onto agarose. Four experiments were conducted to test the effect of the immobilized lipase supplementation on the productive performance of the Japanese quails. During the first 3 weeks, the addition of lipase to poultry diets caused an increase in the body weight gain of birds than the enzyme-free diet. An obvious improvement in quail day egg production during the laying period was observed with the groups fed on a diet supplemented with 3000 and 2000 I U of immobilized lipase per kilogram feed. Blood cholesterol was not affected with lipase addition, while total lipids were significantly increased. Significant reduction was also observed in thyroid hormones (T 3 and T 4 ) as compared with the control group

  6. Incorporation of hydrogel as a sensing medium for recycle of sensing material in chemical sensors

    Science.gov (United States)

    Hwang, Yunjung; Park, Jeong Yong; Kwon, Oh Seok; Joo, Seokwon; Lee, Chang-Soo; Bae, Joonwon

    2018-01-01

    A hydrogel, produced with agarose extracted from seaweed, was introduced as a reusable medium in ultrasensitive sensors employing conducting polymer nanomaterials and aptamers. A basic dopamine (DA) sensor was constructed by placing a hydrogel, containing a sensing material composed of aptamer-linked carboxylated polypyrrole nanotubes (PPy-COOH NTs), onto a micropatterned gold electrode. The hydrogel provided a benign electrochemical environment, facilitated specific interactions between DA and the PPy-COOH NT sensing material, and simplified the retrieval of PPy-COOH NTs after detection. It was demonstrated that the agarose hydrogel was successfully employed as a sensing medium for detection of DA, providing a benign environment for the electrode type sensor. PPy-COOH NTs were recovered by simply heating the hydrogel in water. The hydrogel also afforded stable signal intensity after repeated use with a limit of detection of 1 nmol and a clear, stable signal up to 100 nmol DA. This work provides relevant information for future research on reusable or recyclable sensors.

  7. A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains

    Energy Technology Data Exchange (ETDEWEB)

    Charrier, Thomas; Durand, Marie-Jose; Jouanneau, Sulivan; Thouand, Gerald [UMR CNRS 6144 GEPEA, CBAC, Nantes University, PRES UNAM, Campus de la Courtaisiere-IUT, La Roche-sur-Yon cedex (France); Dion, Michel [UMR CNRS 6204, Nantes University, PRES UNAM, Biotechnologie, Biocatalyse, Bioregulation, 2, Rue de la Houssiniere, BP 92208, Nantes cedex 3 (France); Pernetti, Mimma; Poncelet, Denis [ONIRIS-ENITIAA, UMR CNRS GEPEA, Rue de la Geraudiere, BP 82225, Nantes cedex 3 (France)

    2011-05-15

    This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria-Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30 C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor. (orig.)

  8. A simple and inexpensive method for genomic restriction mapping analysis

    International Nuclear Information System (INIS)

    Huang, C.H.; Lam, V.M.S.; Tam, J.W.O.

    1988-01-01

    The Southern blotting procedure for the transfer of DNA fragments from agarose gels to nitrocellulose membranes has revolutionized nucleic acid detection methods, and it forms the cornerstone of research in molecular biology. Basically, the method involves the denaturation of DNA fragments that have been separated on an agarose gel, the immobilization of the fragments by transfer to a nitrocellulose membrane, and the identification of the fragments of interest through hybridization to /sup 32/P-labeled probes and autoradiography. While the method is sensitive and applicable to both genomic and cloned DNA, it suffers from the disadvantages of being time consuming and expensive, and fragments of greater than 15 kb are difficult to transfer. Moreover, although theoretically the nitrocellulose membrane can be washed and hybridized repeatedly using different probes, in practice, the membrane becomes brittle and difficult to handle after a few cycles. A direct hybridization method for pure DNA clones was developed in 1975 but has not been widely exploited. The authors report here a modification of their procedure as applied to genomic DNA. The method is simple, rapid, and inexpensive, and it does not involve transfer to nitrocellulose membranes

  9. Social motility in african trypanosomes.

    Directory of Open Access Journals (Sweden)

    Michael Oberholzer

    2010-01-01

    Full Text Available African trypanosomes are devastating human and animal pathogens that cause significant human mortality and limit economic development in sub-Saharan Africa. Studies of trypanosome biology generally consider these protozoan parasites as individual cells in suspension cultures or in animal models of infection. Here we report that the procyclic form of the African trypanosome Trypanosoma brucei engages in social behavior when cultivated on semisolid agarose surfaces. This behavior is characterized by trypanosomes assembling into multicellular communities that engage in polarized migrations across the agarose surface and cooperate to divert their movements in response to external signals. These cooperative movements are flagellum-mediated, since they do not occur in trypanin knockdown parasites that lack normal flagellum motility. We term this behavior social motility based on features shared with social motility and other types of surface-induced social behavior in bacteria. Social motility represents a novel and unexpected aspect of trypanosome biology and offers new paradigms for considering host-parasite interactions.

  10. Vascular tissue engineering by computer-aided laser micromachining.

    Science.gov (United States)

    Doraiswamy, Anand; Narayan, Roger J

    2010-04-28

    Many conventional technologies for fabricating tissue engineering scaffolds are not suitable for fabricating scaffolds with patient-specific attributes. For example, many conventional technologies for fabricating tissue engineering scaffolds do not provide control over overall scaffold geometry or over cell position within the scaffold. In this study, the use of computer-aided laser micromachining to create scaffolds for vascular tissue networks was investigated. Computer-aided laser micromachining was used to construct patterned surfaces in agarose or in silicon, which were used for differential adherence and growth of cells into vascular tissue networks. Concentric three-ring structures were fabricated on agarose hydrogel substrates, in which the inner ring contained human aortic endothelial cells, the middle ring contained HA587 human elastin and the outer ring contained human aortic vascular smooth muscle cells. Basement membrane matrix containing vascular endothelial growth factor and heparin was to promote proliferation of human aortic endothelial cells within the vascular tissue networks. Computer-aided laser micromachining provides a unique approach to fabricate small-diameter blood vessels for bypass surgery as well as other artificial tissues with complex geometries.

  11. Apparent diffusion coefficient measurement in a moving phantom simulating linear respiratory motion.

    Science.gov (United States)

    Kwee, Thomas C; Takahara, Taro; Muro, Isao; Van Cauteren, Marc; Imai, Yutaka; Nievelstein, Rutger A J; Mali, Willem P T M; Luijten, Peter R

    2010-10-01

    The aim of this study was to examine the effect of simulated linear respiratory motion on apparent diffusion coefficient (ADC) measurements. Six rectangular test tubes (14 × 92 mm) filled with either water, tomato ketchup, or mayonnaise were positioned in a box containing agarose gel. This box was connected to a double-acting pneumatic cylinder, capable of inducing periodic linear motion in the long-axis direction of the magnetic bore (23-mm stroke). Diffusion-weighted magnetic resonance imaging was performed for both the static and moving phantoms, and ADC measurements were made in the six test tubes in both situations. In the three test tubes whose long axes were parallel to the direction of motion, ADCs agreed well between the moving and static phantom situations. However, in two test tubes that were filled with fluids that had a considerably lower diffusion coefficient than the surrounding agarose gel, and whose long axes were perpendicular to the direction of motion, the ADCs agreed poorly between the moving and static phantom situations. ADC measurements of large homogeneous structures are not affected by linear respiratory motion. However, ADC measurements of inhomogeneous or small structures are affected by linear respiratory motion due to partial volume effects.

  12. Propagating the missing bacteriophages: a large bacteriophage in a new class

    Directory of Open Access Journals (Sweden)

    Hardies Stephen C

    2007-02-01

    Full Text Available Abstract The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly. As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter, tail (486 × 26 nm, corkscrew-like tail fibers (187 × 10 nm and genome (221 Kb that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage, has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.

  13. Growth of magnetite films by a hydrogel method

    International Nuclear Information System (INIS)

    Velásquez, A.A.; Marín, C.C.; Urquijo, J.P.

    2017-01-01

    Magnetite (Fe 3 O 4 ) films were grown on glass substrates by formation and condensation of complex of iron oxides in an agarose hydrogel. The obtained films were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Room Temperature Mössbauer Spectroscopy (TMS), Vibrating Sample Magnetometry (VSM), Atomic Force Microscopy (AFM) and Voltage vs. Current measurements by the four-point method. FTIR and TGA measurements showed that some polymer chains of agarose remain linked to the surface of the magnetic particles of the films after heat treatment. SEM measurements showed that the films are composed by quasi spherical particles with sizes around 55 nm. Mössbauer spectroscopy measurements showed two sextets with broaden lines, which were assigned to magnetite with a distributed particle size, and two doublets, which were assigned to superparamagnetic phases of magnetite. For the specific dimensions of the films prepared, measurements of Voltage vs. Current showed an ohmic behavior for currents between 0 and 200 nA, with a resistance of 355 kΩ.

  14. Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

    Directory of Open Access Journals (Sweden)

    Wensheng Lan

    2012-06-01

    Full Text Available We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol. Briefly, MPH containing six sequential histidines (6× His tag at its N-terminal was bound to nitrilotriacetic acid (NTA agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications.

  15. Growth conditions determine the DNF2 requirement for symbiosis.

    Directory of Open Access Journals (Sweden)

    Fathi Berrabah

    Full Text Available Rhizobia and legumes are able to interact in a symbiotic way leading to the development of root nodules. Within nodules, rhizobia fix nitrogen for the benefit of the plant. These interactions are efficient because spectacularly high densities of nitrogen fixing rhizobia are maintained in the plant cells. DNF2, a Medicago truncatula gene has been described as required for nitrogen fixation, bacteroid's persistence and to prevent defense-like reactions in the nodules. This manuscript shows that a Rhizobium mutant unable to differentiate is not sufficient to trigger defense-like reactions in this organ. Furthermore, we show that the requirement of DNF2 for effective symbiosis can be overcome by permissive growth conditions. The dnf2 knockout mutants grown in vitro on agarose or Phytagel as gelling agents are able to produce nodules fixing nitrogen with the same efficiency as the wild-type. However, when agarose medium is supplemented with the plant defense elicitor ulvan, the dnf2 mutant recovers the fix- phenotype. Together, our data show that plant growth conditions impact the gene requirement for symbiotic nitrogen fixation and suggest that they influence the symbiotic suppression of defense reactions in nodules.

  16. Composition of MRI phantom equivalent to human tissues

    International Nuclear Information System (INIS)

    Kato, Hirokazu; Kuroda, Masahiro; Yoshimura, Koichi; Yoshida, Atsushi; Hanamoto, Katsumi; Kawasaki, Shoji; Shibuya, Koichi; Kanazawa, Susumu

    2005-01-01

    We previously developed two new MRI phantoms (called the CAG phantom and the CAGN phantom), with T1 and T2 relaxation times equivalent to those of any human tissue at 1.5 T. The conductivity of the CAGN phantom is equivalent to that of most types of human tissue in the frequency range of 1 to 130 MHz. In this paper, the relaxation times of human tissues are summarized, and the composition of the corresponding phantoms are provided in table form. The ingredients of these phantoms are carrageenan as the gelling agent, GdCl 3 as a T1 modifier, agarose as a T2 modifier, NaCl (CAGN phantom only) as a conductivity modifier, NaN 3 as an antiseptic, and distilled water. The phantoms have T1 values of 202-1904 ms and T2 values of 38-423 ms when the concentrations of GdCl 3 and agarose are varied from 0-140 μmol/kg, and 0%-1.6%, respectively, and the CAGN phantom has a conductivity of 0.27-1.26 S/m when the NaCl concentration is varied from 0%-0.7%. These phantoms have sufficient strength to replicate a torso without the use of reinforcing agents, and can be cut by a knife into any shape. We anticipate the CAGN phantom to be highly useful and practical for MRI and hyperthermia-related research

  17. Binding analysis of ferritin with heme using α-casein and biotinylated-hemin: detection of heme-binding capacity of Dpr derived from heme synthesis-deficient Streptococcus mutans.

    Science.gov (United States)

    Mieno, Ayako; Yamamoto, Yuji; Yoshikawa, Yasunaga; Watanabe, Kiyotaka; Mukai, Takao; Orino, Koichi

    2013-01-01

    Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to an α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Escherichia coli, did not bind to an α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After preincubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin and that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.

  18. A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals: application to the streptavidin/biotin system

    International Nuclear Information System (INIS)

    Dunn-Dufault, Robert; Pollak, Alfred; Fitzgerald, Jane; Thornback, John R.; Ballinger, James R.

    2000-01-01

    A high effective specific activity (HESA) formulation of a biotin-containing 99m Tc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 was coupled to a maleimide-functionalized polyethylene glycol resin via a thiol ether linkage and labeled with 99m Tc-gluconate at room temperature, followed by elution of the HESA 99m Tc-RP488 in saline (minimum specific activity ∼ 1000 TBq/mmol by amino acid analysis). Both HESA and LESA 99m Tc-RP488 labeled at > 90% purity. In vitro, HESA 99m Tc-RP488 incubated with streptavidin-agarose was bound quantitatively, but there was competition from addition of increasing amounts of cold RP488. In rats, radiotracer uptake was evident at the site of implantation of streptavidin-agarose beads for the HESA dose, less uptake of low effective specific activity (LESA) material, and no appreciable uptake in the control rats of the LESA or HESA dose. The target-to-background ratio for HESA 99m Tc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting 99m Tc radiopharmaceuticals

  19. Attempt to run urinary protein electrophoresis using capillary technique.

    Science.gov (United States)

    Falcone, Michele

    2014-10-01

    The study of urinary protein has a predominant place in the diagnosis of kidney disease. The most common technique is agarose gel electrophoresis (AGE). For several years, the technique of choice applied to the analysis of serum proteins has been CE, a system that uses capillary fused silica, subjected to high voltage to separate and measure serum proteins. The purpose of this paper was to perform capillary electrophoresis on urinary proteins which, at present, are not interpretable due to the many nonspecific peaks visible when using gel electrophoresis. In order to carry out our research, we used a capillary V8 analyzer together with an agarose gel system from the same company. AGE was taken as the reference method, for which urine was used without any pretreatment. For the V8 system, urine was subjected to purification on granular-activated carbon and then inserted into the V8 analyzer, selecting a program suitable for liquids with low protein content. We examined 19 urine samples collected over 24 hrs from both hospitalized and external patients with different types of proteinuria plus a serum diluted 1/61 considered as a control to recognize the bands. Both methods showed the same protein fractions and classified the proteinuria in a similar way. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Determination of size distribution of small DNA fragments by polyacrylamide gel electrophoresis

    International Nuclear Information System (INIS)

    Lau How Mooi

    1998-01-01

    Size distribution determination of DNA fragments can be normally determined by the agarose gel electrophoresis, including the normal DNA banding pattern analysis. However this method is only good for large DNA, such as the DNA of the size of kilo base pairs to mega base pairs range. DNA of size less than kilo base pairs is difficult to be quantified by the agarose gel method. Polyacrylamide gel electrophoresis however can be used to measure the quantity of DNA fragments of size less than kilo base pairs in length, down to less than ten base pairs. This method is good for determining the quantity of the smaller size DNA, single stranded polymers or even some proteins, if the known standards are available. In this report detail description of the method of preparing the polyacrylamide gel, and the experimental set up is discussed. Possible uses of this method, and the comparison with the standard sizes of DNA is also shown. This method is used to determine the distribution of the amount of the fragmented DNA after the Calf-thymus DNA has been exposed to various types of radiation and of different doses. The standards were used to determine the sizes of the fragmented Calf-thymus DNA. The higher the dose the higher is the amount of the smaller size DNA measured

  1. Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

    Science.gov (United States)

    Mikaeili, F; Kia, E B; Sharbatkhori, M; Sharifdini, M; Jalalizand, N; Heidari, Z; Zarei, Z; Stensvold, C R; Mirhendi, H

    2013-06-01

    Six simple methods for extraction of ribosomal and mitochondrial DNA from Toxocara canis, Toxocara cati and Toxascaris leonina were compared by evaluating the presence, appearance and intensity of PCR products visualized on agarose gels and amplified from DNA extracted by each of the methods. For each species, two isolates were obtained from the intestines of their respective hosts: T. canis and T. leonina from dogs, and T. cati from cats. For all isolates, total DNA was extracted using six different methods, including grinding, boiling, crushing, beating, freeze-thawing and the use of a commercial kit. To evaluate the efficacy of each method, the internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit 1 (cox1) gene were chosen as representative markers for ribosomal and mitochondrial DNA, respectively. Among the six DNA extraction methods, the beating method was the most cost effective for all three species, followed by the commercial kit. Both methods produced high intensity bands on agarose gels and were characterized by no or minimal smear formation, depending on gene target; however, beating was less expensive. We therefore recommend the beating method for studies where costs need to be kept at low levels. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Neurospora ribosomal DNA sequences are indistinguishable within cell types but distinguishable among heterothallic species

    International Nuclear Information System (INIS)

    Chambers, C.; Dutta, S.K.

    1983-01-01

    High molecular nuclear DNAs were isolated from three developmental cell types of N. crassa: conidia, mycelia and germinated conidia, and from mycelial cells of two other heterothallic species, N. intermedia and N. sitophila. These nuclear DNAs were treated with several restriction enzymes: EcoR1, Bam H1, Hind III, Hinc II, Bgl II, Sma I and Pst 1. All seven restriction enzymes were tested on 0.7% agarose gels. EcoR1, Hind III, Pst 1, and Hinc II showed band differences among the species, but not among the cell types. Southern blot transfers of restricted DNA gels were then hybridized with 32 P-labelled pMF2 rDNAs (probe). This later DNA was prepared from N. crassa rDNA cloned into pBR322 plasmid, obtained from Dr. Robert Metzenberg of the University of Wisconsin. Autoradiograms of these hybrids between southern blots and probe DNA revealed similar rDNA band patterns confirming the observations on restriction gels. In the case of EcoR1 restriction analysis there were differences in fragments on 0.7% agarose gel, but after hybridization of southern blots no differences in band patterns were seen in autoradiograms. This raises the question whether the background bands were all of rDNA sequences. These studies are being continued using ITS (internal transcribed spacer) sequences of N. crassa rDNAs cloned in pBR322 plasmid

  3. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

  4. Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

    Science.gov (United States)

    Choi, Hoseok; Choi, Bomi; Seo, Ju Tae; Lee, Kyung Jin; Gye, Myung Chan; Kim, Young-Pil

    2016-01-01

    Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts. PMID:27092510

  5. Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium

    International Nuclear Information System (INIS)

    Sandru, G.; Greiner, R.

    1994-01-01

    Incorporation of [ 3 H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulated significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 6 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures. 42 refs., 9 figs., 3 tabs

  6. Isolation of a mannose/N-acetylglucosamine receptor from rabbit lung

    International Nuclear Information System (INIS)

    Lennartz, M.R.; Wileman, T.E.; Stahl, P.D.

    1986-01-01

    The presence of a mannose receptor on alveolar macrophages was first described in 1978 and later extended to other macrophage populations. Recently the novel ligand, mannose-conjugated lactoperoxidase, was used to identify this receptor as a 175kD protein. A 175kD protein exhibiting mannose and N-acetylglucosamine (GlcNAc)-binding properties was isolated from rabbit lung membranes. Membranes were washed with high salt, mannose and EDTA to remove endogenously bound ligand and were subsequently extracted with 1% Triton-X 100. The extract was subjected to affinity chromatography on Mannose-Sepharose followed by GlcNAc-Agarose. Triton was exchanged for 1% CHAPS while the protein was bound to GlcNAc-Agarose, allowing the eluate to be concentrated without denaturation. The eluted protein bound [ 125 I]mannose-BSA in a mannan-inhibitable fashion. Microgram quantities of protein were isolated in this fashion. SDS-PAGE revealed a major protein band at 175kD. Amino acid analysis indicates low concentrations of methionine. Results from concanavalin A binding studies and endoglycosidase F digestion suggest that the mannose receptor is a glycoprotein containing N-linked oligosaccharides

  7. [Fundamental evaluation of apolipoprotein B-48 by chemiluminescence enzyme immunoassay--identification of apolipoprotein B-48 with immunoblotting].

    Science.gov (United States)

    Sato, Itsuko; Fujioka, Yoshio; Hayashi, Fujio; Mukai, Masahiko; Kawano, Seiji; Ishikawa, Yuichi; Yamashita, Shizuya; Kumagai, Shunichi

    2007-06-01

    Apolipoprotein B-48 (apo B-48) is a constituent of chylomicrons and chylomicron remnants, and its fasting concentration has been reported to be a marker of postprandial hyperlipidemia, which is thought to be a risk factor of atherosclerosis. We evaluated the serum apo B-48 concentrations by chemiluminescence enzyme immunoassay (CLEIA), which was recently introduced as Lumipulse f fully automated immunosaasy analyzer by Fujirebio Inc (Tokyo, Japan), and performed immunoblotting on agarose gel electrophoresis with anti-apo B-48 antibody. Apo B-48 assay was intra-assay reproducible (CVs: 1.9-3.1%) and inter-assay reproducible (CVs: 2.2-4.4%). The assay range for apo B-48 was from 0.2 to 40.0 microg/ml. The effects of interfering substances such as free/conjugated birirubin, hemoglobin, Intrafat, ascorbic acid and rheumatoid factor were negligible. For storage, it was preferable to freeze, and to avoid frozen-thaw process as much as possible. Anti-apo B-48 antibody was reactive over a wide range from origin to the position of very-low-density lipoproteins in immunoblotting after agarose gel electrophoresis. Apo B-48 measurement by CLEIA was feasible to clinical use for the assessment of lipoprotein metabolism.

  8. Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

    Science.gov (United States)

    Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

    2012-01-01

    We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

  9. Bimodal cell death induced by high radiation doses in the radioresistant sf9 insect cell line

    International Nuclear Information System (INIS)

    Chandna, S.

    2003-01-01

    Full text: This study was conducted to investigate the mode(s) of cell death induced by high radiation doses in the highly radioresistant Sf9 insect ovarian cell line. Methods: Cells were exposed to γ-radiation doses 200Gy and 500Gy, harvested at various time intervals (6h-72h) following irradiation, and subjected to cell morphology assay, DNA agarose gel electrophoresis, single cell gel electrophoresis (SCGE; comet assay) and Annexin-V labeling for the detection of membrane phosphatidylserine externalization. Cell morphology was assessed in cells entrapped and fixed in agarose gel directly from the cell suspension, thus preventing the possible loss of fragments/ apoptotic bodies. Surviving fraction of Sf9 cells was 0.01 at 200Gy and 98%) undergoing extensive DNA fragmentation at 500Gy, whereas the frequency of cells with DNA fragmentation was considerably less (∼12%) at 200Gy. Conclusions: While the mode of cell death at 200Gy seems to be different from typical apoptosis, a dose of 500Gy induced bimodal cell death, with typical apoptotic as well as the atypical cell death observed at 200Gy

  10. Sequence Dependent Electrophoretic Separations of DNA in Pluronic F127 Gels

    Science.gov (United States)

    You, Seungyong; van Winkle, David H.

    2010-03-01

    Two-dimensional (2-D) electrophoresis has successfully been used to visualize the separation of DNA fragments of the same length. We electrophorese a double-stranded DNA ladder in an Agarose gel for the first dimension and in gels of Pluronic F127 for the second dimension at room temperature. The 1000 bp band that travels together as a single band in an Agarose gel is split into two bands in Pluronic gels. The slower band follows the exponential decay trend that the other ladder constituents do. After sequencing the DNA fragments, the faster band has an apparently random sequence, while the slower band and the others have two A-tracts in each 250 bp segment. The A-tracts consist of a series of at least five adenine bases pairing with thymine bases. This result leads to the conclusion that the migration of the DNA molecules bent with A-tracts is more retarded in Pluronic gels than the wild-type of DNA molecules.

  11. An easy to produce and economical three-dimensional brain phantom for stereotactic computed tomographic-guided brain biopsy training in the dog.

    Science.gov (United States)

    Sidhu, Deepinder S; Ruth, Jeffrey D; Lambert, Gregory; Rossmeisl, John H

    2017-07-01

    To develop and validate a three-dimensional (3D) brain phantom that can be incorporated into existing stereotactic headframes to simulate stereotactic brain biopsy (SBB) and train veterinary surgeons. Experimental study. Canine brain phantoms were fabricated from osteological skull specimens, agarose brain parenchyma, and cheddar and mozzarella cheese molds (simulating meningiomas and gliomas). The neuroradiologic and viscoelastic properties of phantoms were quantified with computed tomography (CT) and oscillatory compression tests, respectively. Phantoms were validated by experienced and novice operators performing SBB on phantoms containing randomly placed, focal targets. Target yield and needle placement error (NPE) were compared between operators. Phantoms were produced in brain parenchyma, and contrast-enhancing tumors of meningeal and glial origin, respectively. The complex moduli of the agarose and cheeses were comparable to the viscoelastic properties of in vivo brain tissues and brain tumors. The overall diagnostic yield of SBB was 88%. Although NPE did not differ between novice (median 3.68 mm; range, 1.46-14.54 mm) and experienced surgeons (median 1.17 mm, range, 0.78-1.58 mm), our results support the relevance of the learning curve associated with the SBB procedure. This 3D phantom replicates anatomical, CT, and tactile features of brain tissues and tumors and can be used to develop the technical skills required to perform SBB. © 2017 The American College of Veterinary Surgeons.

  12. Purification and identification of the fusicoccin binding protein from oat root plasma membrane

    Science.gov (United States)

    de Boer, A. H.; Watson, B. A.; Cleland, R. E.

    1989-01-01

    Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

  13. Low buoyant density proteoglycans from saline and dissociative extracts of embryonic chicken retinas

    Energy Technology Data Exchange (ETDEWEB)

    Morris, J.E.; Ting, Y.P.; Birkholz-Lambrecht, A.

    1984-03-01

    Retinas were labeled in culture with (/sup 3/H)glucosamine or (/sup 3/H)leucine and (/sup 35/S)sulfate and extracted sequentially with physiologically balanced saline and 4 M guanidine HCl. They were dialyzed into associative conditions (0.5 M NaCl) and chromatographed on agarose columns. Under these conditions, some of the proteoglycans were associated in massive complexes that showed low buoyant densities when centrifuged in CsCl density gradients under dissociative conditions (4 M guanidine HCl). Much of the label in these complexes was in molecules other than proteoglycans. Most of the proteoglycans, however, were included on the agarose columns, where they appeared to be constitutionally of low buoyant density. They resisted attempts to separate potential low buoyant density contaminants from the major proteoglycans by direct CsCl density gradient centrifugation or by the fractionation of saline or 8 M urea extracts on diethylaminoethyl-Sephacel. The diethylaminoethyl-Sephacel fractions were either subjected to CsCl density gradient centrifugation or were chromatographed on Sephacryl S-300, in both cases before and after alkaline cleavage, to confirm the presence of typical O-linked glycosaminoglycans. The medium and balanced salt extracts were enriched in chondroitin sulfate and other sulfated macromolecules, possibly highly sulfated oligosaccharides, that resisted digestion by chondroitinase ABC but were electrophoretically less mobile than heparan sulfate. Guanidine HCl or urea extracts of the residues were mixtures of high and low density proteoglycans that were enriched in heparan sulfate.

  14. Metal artefact reduction in MRI at both 1.5 and 3.0 T using slice encoding for metal artefact correction and view angle tilting

    Science.gov (United States)

    Reichert, M; Morelli, J N; Nittka, M; Attenberger, U; Runge, V M

    2015-01-01

    Objective: To compare metal artefact reduction in MRI at both 3.0 T and 1.5 T using different sequence strategies. Methods: Metal implants of stainless steel screw and plate within agarose phantoms and tissue specimens as well as three patients with implants were imaged at both 1.5 T and 3.0 T, using view angle tilting (VAT), slice encoding for metal artefact correction with VAT (SEMAC-VAT) and conventional sequence. Artefact reduction in agarose phantoms was quantitatively assessed by artefact volume measurements. Blinded reads were conducted in tissue specimen and human imaging, with respect to artefact size, distortion, blurring and overall image quality. Wilcoxon and Friedman tests for multiple comparisons and intraclass correlation coefficient (ICC) for interobserver agreement were performed with a significant level of p 3.0 T (p 3.0 T. Advances in knowledge: The feasibility of metal artefact reduction with SEMAC-VAT was demonstrated at 3.0-T MR. SEMAC-VAT significantly reduced metal artefacts at both 1.5 and 3.0 T. SEMAC-VAT allowed for better visualization of the tissue structures adjacent to the metal implants. SEMAC-VAT produced consistently better image quality in both tissue specimen and human imaging. PMID:25613398

  15. Functional and Physical Outcomes following Use of a Flexible CO2 Laser Fiber and Bipolar Electrocautery in Close Proximity to the Rat Sciatic Nerve with Correlation to an In Vitro Thermal Profile Model

    Directory of Open Access Journals (Sweden)

    A. M. Robinson

    2015-01-01

    Full Text Available This study compared functional and physical collateral damage to a nerve when operating a Codman MALIS Bipolar Electrosurgical System CMC-III or a CO2 laser coupled to a laser, with correlation to an in vitro model of heating profiles created by the devices in thermochromic ink agarose. Functional damage of the rat sciatic nerve after operating the MALIS or CO2 laser at various power settings and proximities to the nerve was measured by electrically evoked nerve action potentials, and histology of the nerve was used to assess physical damage. Thermochromic ink dissolved in agarose was used to model the spatial and temporal profile of the collateral heating zone of the electrosurgical system and the laser ablation cone. We found that this laser can be operated at 2 W directly above the nerve with minimal damage, while power settings of 5 W and 10 W resulted in acute functional and physical nerve damage, correlating with the maximal heating cone in the thermochromic ink model. MALIS settings up to 40 (11 W did not result in major functional or physical nerve damage until the nerve was between the forceps tips, correlating with the hottest zone, localized discretely between the tips.

  16. Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

    Science.gov (United States)

    Park, Ji Eun; Park, Min Hee; Kim, Min Seong; Park, Yeo Reum; Yun, Jung Im; Cheong, Hee Tae; Kim, Minseok; Choi, Jung Hoon; Lee, Eunsong; Lee, Seung Tae

    2017-12-01

    Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research. © 2017 International Federation for Cell Biology.

  17. Manganese modified CdTe/CdS quantum dots as an immunoassay biosensor for the detection of Golgi protein-73.

    Science.gov (United States)

    Liu, Wei; Zhang, Aixia; Xu, Guanhong; Wei, Fangdi; Yang, Jing; Hu, Qin

    2016-01-05

    In this paper, a new fluorescence bioassay for Golgi protein-73 (GP73), a promising marker for monitoring liver tumor, was developed by using anti-GP73 antibody (GP73 Ab) capped quantum dots (QDs) coupled with protein A/G agarose beads in an attempt to improve the analysis time, cost and operation. First, carboxylic-functionalized Mn modified CdTe/CdS QDs were synthesized and covalently conjugated with GP73 Ab, then protein A/G agarose beads were specifically combined with the QDs-conjugated Ab to form the QDs-Ab-beads conjugate, which could capture and separate GP73 from the sample through simple centrifugation. It was found that the fluorescence intensity of the above QDs-Ab-beads biosensor could be specifically quenched by GP73 added. A simple, rapid and specific quantitative method for GP73 protein was proposed using the as-prepared QDs-Ab-beads as a biosensor. Under the optimized conditions, the calibration curve of the proposed assay showed good linearity with a correlation coefficient of 0.9935 in the concentration range of 20-150 ng/mL of GP73 protein. The limit of detection (defined as 3σ/K) was 10 ng/mL. The method built exhibited a great potential in the clinic test of GP73. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Apparent diffusion coefficient measurement in a moving phantom simulating linear respiratory motion

    International Nuclear Information System (INIS)

    Kwee, T.C.; Takahara, Taro; Nievelstein, R.A.J.; Mali, W.P.T.M.; Luijten, P.R.; Muro, Isao; Imai, Yutaka; Cauteren, M. Van

    2010-01-01

    The aim of this study was to examine the effect of simulated linear respiratory motion on apparent diffusion coefficient (ADC) measurements. Six rectangular test tubes (14 x 92 mm) filled with either water, tomato ketchup, or mayonnaise were positioned in a box containing agarose gel. This box was connected to a double-acting pneumatic cylinder, capable of inducing periodic linear motion in the long-axis direction of the magnetic bore (23-mm stroke). Diffusion-weighted magnetic resonance imaging was performed for both the static and moving phantoms, and ADC measurements were made in the six test tubes in both situations. In the three test tubes whose long axes were parallel to the direction of motion, ADCs agreed well between the moving and static phantom situations. However, in two test tubes that were filled with fluids that had a considerably lower diffusion coefficient than the surrounding agarose gel, and whose long axes were perpendicular to the direction of motion, the ADCs agreed poorly between the moving and static phantom situations. ADC measurements of large homogeneous structures are not affected by linear respiratory motion. However, ADC measurements of inhomogeneous or small structures are affected by linear respiratory motion due to partial volume effects. (author)

  19. In vitro incorporation of radiolabeled cholesteryl esters into high and low density lipoproteins

    International Nuclear Information System (INIS)

    Terpstra, A.H.; Nicolosi, R.J.; Herbert, P.N.

    1989-01-01

    We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL). Radiolabeled cholesteryl esters dissolved in absolute ethanol were mixed with LDL or HDL in the presence of lipoprotein-deficient serum (LPDS) as a source of core lipid transfer activity. The efficiency of incorporation was dependent on: (a) the core lipid transfer activity and quantity of LPDS, (b) the mass of added radiolabeled cholesteryl esters, (c) the length of incubation, and (d) the amount of acceptor lipoprotein cholesterol. The tracer incorporation was documented by repeat density gradient ultracentrifugation, agarose gel electrophoresis, and precipitation with heparin-MnCl2. The radiolabeling conditions did not affect the following properties of the lipoproteins: (1) chemical composition, (2) electrophoretic mobility on agarose gels, (3) hydrated density, (4) distribution of apoproteins on SDS gels, (5) plasma clearance rates, and (6) immunoprecipitability of HDL apoproteins A-I and A-II. Rat HDL containing radiolabeled cholesteryl esters incorporated in vitro had plasma disappearance rates identical to HDL radiolabeled in vivo

  20. Electrochemical writing on edible polysaccharide films for intelligent food packaging.

    Science.gov (United States)

    Wu, Si; Wang, Wenqi; Yan, Kun; Ding, Fuyuan; Shi, Xiaowen; Deng, Hongbing; Du, Yumin

    2018-04-15

    Polysaccharide films used as intelligent food packaging possess the advantages of renewability, safety and biodegradability. Printing on the polysaccharidic food packaging is challenging due to the high demand for edible-ink and the need for a suitable printing technique. In this work, we propose an electrochemical method for writing on polysaccharide film. Unlike conventional printing, this electrochemical writing process relies on the pH responsive color change of anthocyanin embedded in the chitosan/agarose hydrogel. By biasing a negative potential to a stainless wire (used as a pen) contacting the surface of the chitosan/agarose/ATH hydrogel, the locally generated pH change induced the color change of ATH and wrote programmed information on the hydrogel. We demonstrate the writing can be temporary in the hydrogel but stable when the hydrogel is dried. We further demonstrate that the written film is applicable for the detection of the spoilage of crucian fish. The reported electrochemical writing process provides a novel method for printing information on polysaccharide film and great potential for intelligent food packaging. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. A Repeating Sulfated Galactan Motif Resuscitates Dormant Micrococcus luteus Bacteria.

    Science.gov (United States)

    Böttcher, Thomas; Szamosvári, Dávid; Clardy, Jon

    2018-07-01

    Only a small fraction of bacteria can autonomously initiate growth on agar plates. Nongrowing bacteria typically enter a metabolically inactive dormant state and require specific chemical trigger factors or signals to exit this state and to resume growth. Micrococcus luteus has become a model organism for this important yet poorly understood phenomenon. Only a few resuscitation signals have been described to date, and all of them are produced endogenously by bacterial species. We report the discovery of a novel type of resuscitation signal that allows M. luteus to grow on agar but not agarose plates. Fractionation of the agar polysaccharide complex and sulfation of agarose allowed us to identify the signal as highly sulfated saccharides found in agar or carrageenans. Purification of hydrolyzed κ-carrageenan ultimately led to the identification of the signal as a small fragment of a large linear polysaccharide, i.e., an oligosaccharide of five or more sugars with a repeating disaccharide motif containing d-galactose-4-sulfate (G4S) 1,4-linked to 3,6-anhydro-α-d-galactose (DA), G4S-(DA-G4S) n ≥2 IMPORTANCE Most environmental bacteria cannot initiate growth on agar plates, but they can flourish on the same plates once growth is initiated. While there are a number of names for and manifestations of this phenomenon, the underlying cause appears to be the requirement for a molecular signal indicating safe growing conditions. Micrococcus luteus has become a model organism for studying this growth initiation process, often called resuscitation, because of its apparent connection with the persistent or dormant form of Mycobacterium tuberculosis , an important human pathogen. In this report, we identify a highly sulfated saccharide from agar or carrageenans that robustly resuscitates dormant M. luteus on agarose plates. We identified and characterized the signal as a small repeating disaccharide motif. Our results indicate that signals inherent in or absent from the

  2. Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

    Directory of Open Access Journals (Sweden)

    Ciervo Alessandra

    2006-04-01

    Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

  3. Standardizing electrophoresis conditions: how to eliminate a major source of error in the comet assay.

    Directory of Open Access Journals (Sweden)

    Gunnar Brunborg

    2015-06-01

    Full Text Available In the alkaline comet assay, cells are embedded in agarose, lysed, and then subjected to further processing including electrophoresis at high pH (>13. We observed very large variations of mean comet tail lengths of cell samples from the same population when spread on a glass or plastic substrate and subjected to electrophoresis. These variations might be cancelled out if comets are scored randomly over a large surface, or if all the comets are scored. The mean tail length may then be representative of the population, although its standard error is large. However, the scoring process often involves selection of 50 – 100 comets in areas selected in an unsystematic way from a large gel on a glass slide. When using our 96-sample minigel format (1, neighbouring sample variations are easily detected. We have used this system to study the cause of the comet assay variations during electrophoresis and we have defined experimental conditions which reduce the variations to a minimum. We studied the importance of various physical parameters during electrophoresis: (i voltage; (ii duration of electrophoresis; (iii electric current; (iv temperature; and (v agarose concentration. We observed that the voltage (V/cm varied substantially during electrophoresis, even within a few millimetres of distance between gel samples. Not unexpectedly, both the potential ( V/cm and the time were linearly related to the mean comet tail, whereas the current was not. By measuring the local voltage with microelectrodes a few millimetres apart, we observed substantial local variations in V/cm, and they increased with time. This explains the large variations in neighbouring sample comet tails of 25% or more. By introducing simple technology (circulation of the solution during electrophoresis, and temperature control, these variations in mean comet tail were largely abolished, as were the V/cm variations. Circulation was shown to be particularly important and optimal conditions

  4. Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

    Directory of Open Access Journals (Sweden)

    Hideyuki Arimitsu

    Full Text Available Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e, a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

  5. Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses.

    Science.gov (United States)

    Strong, Averey D; Daniels, Richard L

    2017-08-02

    The tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2. GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student's t-tests and one-way ANOVA. We found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells. We demonstrate the use

  6. High index glass thin film processing for photonics and photovoltaic (PV) applications

    Science.gov (United States)

    Ogbuu, Okechukwu Anthony

    To favorably compete with fossil-fuel technology, the greatest challenge for thin film solar-cells is to improve efficiency and reduce material cost. Thickness scaling to thin film reduces material cost but affects the light absorption in the cells; therefore a concept that traps incident photons and increases its optical path length is needed to boost absorption in thin film solar cells. One approach is the integration of low symmetric gratings (LSG), using high index material, on either the front-side or backside of 30 um thin c-Si cells. In this study, Multicomponent TeO2--Bi2O 3--ZnO (TBZ) glass thin films were prepared using RF magnetron sputtering under different oxygen flow rates. The influences of oxygen flow rate on the structural and optical properties of the resulting thin films were investigated. The structural origin of the optical property variation was studied using X-ray diffraction, X-ray photoelectron spectroscopy, Raman Spectroscopy, and transmission electron microscopy. The results indicate that TBZ glass thin film is a suitable material for front side LSG material photovoltaic and photonics applications due to their amorphous nature, high refractive index (n > 2), broad band optical transparency window, low processing temperature. We developed a simple maskless method to pattern sputtered tellurite based glass thin films using unconventional agarose hydrogel mediated wet etching. Conventional wet etching process, while claiming low cost and high throughput, suffers from reproducibility and pattern fidelity issues due to the isotropic nature of wet chemical etching when applied to glasses and polymers. This method overcomes these challenges by using an agarose hydrogel stamp to mediate a conformal etching process. In our maskless method, agarose hydrogel stamps are patterned following a standard soft lithography and replica molding process from micropatterned masters and soaked in a chemical etchant. The micro-scale features on the stamp are

  7. [Ulysses retrotransposon aspartate proteinase (Drosophila virilis)].

    Science.gov (United States)

    Volkov, D A; Savvateeva, L V; Dergousova, N I; Rumsh, L D

    2002-01-01

    Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

  8. New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

    Science.gov (United States)

    Volkov, D A; Dergousova, N I; Rumsh, L D

    2004-06-01

    This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

  9. Application of a rapid screening method to detect irradiated meat in Brazil

    International Nuclear Information System (INIS)

    Villavicencio, A.L.C.H.; Delincee, H.

    1998-01-01

    Complete text of publication follows. Based on the enormous potential for food irradiation in Brazil, and to ensure free consumer choice, there is a need to find a convenient and rapid method for detection of irradiated food. Since treatment with ionizing radiation causes DNA fragmentation, the analysis of DNA damage might be promising. In fact, DNA fragmentation measured in single cells by agarose gel electrophoresis - DNA Comet Assay - has shown to offer great potential as a rapid tool to detect whether a wide variety of foodstuffs has been radiation processed. However, more work is needed to exploit the full potential of this promising technique. In this paper, the DNA Comet Assay was used to identify exotic meat (boar, jacare and capybara), irradiated with 60 Co gamma-rays. The applied radiation doses were 0, 1.5, 3.0 and 4.5 kGy. Analysis of the DNA migration enable a rapid identification of the radiation treatment

  10. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    Directory of Open Access Journals (Sweden)

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

  11. Genomic and gene variation in Mycoplasma hominis strains

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Andersen, H; Birkelund, Svend

    1987-01-01

    DNAs from 14 strains of Mycoplasma hominis isolated from various habitats, including strain PG21, were analyzed for genomic heterogeneity. DNA-DNA filter hybridization values were from 51 to 91%. Restriction endonuclease digestion patterns, analyzed by agarose gel electrophoresis, revealed...... no identity or cluster formation between strains. Variation within M. hominis rRNA genes was analyzed by Southern hybridization of EcoRI-cleaved DNA hybridized with a cloned fragment of the rRNA gene from the mycoplasma strain PG50. Five of the M. hominis strains showed identical hybridization patterns....... These hybridization patterns were compared with those of 12 other mycoplasma species, which showed a much more complex band pattern. Cloned nonribosomal RNA gene fragments of M. hominis PG21 DNA were analyzed, and the fragments were used to demonstrate heterogeneity among the strains. A monoclonal antibody against...

  12. Filaggrin compound heterozygous patients carry mutations in trans position

    DEFF Research Database (Denmark)

    Carlsen, Berit C; Meldgaard, Michael; Johansen, Jeanne D

    2013-01-01

    by means of allele-specific PCR amplification and analysis of PCR products by agarose gel electrophoresis. All R501X/2282del4 compound heterozygous samples collected over a 4-year period of routine FLG mutation testing were investigated. In total, 37 samples were tested. All thirty-seven R501X/2282del4......More than 40 null mutations in the filaggrin (FLG) gene are described. It is therefore possible to find two different null mutations in one individual (compound heterozygosity). It has been generally perceived that homozygous and compound heterozygous individuals were genotypically comparable......; however, this has not been scientifically investigated. Two different FLG null mutations in the same individual may be in trans position, meaning that each mutation locates to a different allele functionally equivalent to homozygosity, or may be in cis position, meaning that both mutations locate...

  13. Activation of Stat-3 is involved in the induction of apoptosis after ligation of major histocompatibility complex class I molecules on human Jurkat T cells

    DEFF Research Database (Denmark)

    Skov, S; Nielsen, M; Bregenholt, S

    1998-01-01

    Activation of Janus tyrosine kinases (Jak) and Signal transducers and activators of transcription (Stat) after ligation of major histocompatibility complex class I (MHC-I) was explored in Jurkat T cells. Cross-linking of MHC-I mediated tyrosine phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3......-probe derived from the interferon-gamma activated site (GAS) in the c-fos promoter, a common DNA sequence for Stat protein binding. An association between hSIE and Stat-3 after MHC-I ligation was directly demonstrated by precipitating Stat-3 from nuclear extracts with biotinylated hSIE probe and avidin......-coupled agarose. To investigate the function of the activated Stat-3, Jurkat T cells were transiently transfected with a Stat-3 isoform lacking the transactivating domain. This dominant-negative acting Stat-3 isoform significantly inhibited apoptosis induced by ligation of MHC-I. In conclusion, our data suggest...

  14. Rapid tryptic mapping using enzymatically active mass spectrometer probe tips

    Energy Technology Data Exchange (ETDEWEB)

    Dogruel, D.; Williams, P.; Nelson, R.W. [Arizona State Univ., Tempe, AZ (United States)

    1995-12-01

    A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. 19 refs., 6 figs., 2 tabs.

  15. Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

    Science.gov (United States)

    Duarte, José M; Barbier, Içvara; Schaerli, Yolanda

    2017-11-17

    Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

  16. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

    Directory of Open Access Journals (Sweden)

    Brigitte Mack

    Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

  17. Identification of new isolates of Bacillus thuringiensis using rep-PCR products and delta-endotoxin electron microscopy

    Directory of Open Access Journals (Sweden)

    A.S.G. Lima

    2002-01-01

    Full Text Available PCR has been used to analyze the distribution of REP (Repetitive Extragenic Palindromic and ERIC (Enterobacterial Repetitive Intergenic Consensus sequences (rep-PCR found within the genome of the bacterium Bacillus thuringiensis, with the purpose to analyze the genetic similarities among 56 subspecies samples and 95 field isolates. The PCR products were analyzed by EB-AGE (ethidium bromide-agarose electrophoresis and then submitted to banding comparisons, based on the Phyllip software algorithm. When the banding similarities were considered for comparison purposes among all the strains, the phylogenic tree patterns varied according to the rep-PCR primers considered, but, from a broader point of view, the ERIC sequences produced better results, which, together with electron microscopy analysis of the released parasporal bodies and colony morphology characteristics, allowed to detect two possible new subspecies of B. thuringiensis.

  18. RFLP for Duchenne muscular dystrophy cDNA clone 30-2

    Energy Technology Data Exchange (ETDEWEB)

    Walker, A P; Bartlett, R J; Laing, N G; Siddique, T; Yamaoka, L H; Chen, J C; Hung, W Y; Roses, A D [Duke Univ. Medical Center, Durham, NC (USA)

    1988-09-26

    30-2 is one of 6 cDNA clones which comprise the cDNA for the Duchenne muscular dystrophy gene. It is a 1.15 kb fragment in the EcoRI site of Bluescribe. TaqI (T{down arrow}CGA) identifies two bands with alleles at 3.7 and 3.5 kb, as well as eight constant bands at 9.0, 7.5, 4.6, 3.6, 3.4, 2.5, 1.7 and 1.4 kb. The allele frequency was studied in 47 unrelated DMD males: 3.7 kb allele 0.45; and 3.5 kb allele 0.55. Co-dominant X-linked segregation was demonstrated in two 2-generation families. 1.1% agarose gels required to resolve the bands. The polymorphism is also recognized by PERT 87-15.

  19. HLA-DPB1 typing with polymerase chain reaction and restriction fragment length polymorphism technique in Danes

    DEFF Research Database (Denmark)

    Hviid, Thomas Vauvert F.; Madsen, Hans O; Morling, Niels

    1992-01-01

    We have used the polymerase chain reaction (PCR) in combination with the restriction fragment length polymorphism (RFLP) technique for HLA-DBP1 typing. After PCR amplification of the polymorphic second exon of the HLA-DPB1 locus, the PCR product was digested with seven allele-specific restriction...... endonucleases: RsaI, FokI, ApaI, SacI, BstUI, EcoNI, and DdeI, and the DNA fragments were separated by electrophoresis in agarose gels. Altogether, 71 individuals were investigated and 16 different HLA-DPB1 types were observed in 26 different heterozygotic combinations, as well as five possible homozygotes....... Four heterozygotes could not be unequivocally typed with the PCR-RFLP method. The HLA-DPB1 typing results obtained with the PCR-RFLP method were compared with the typing results obtained with PCR allele-specific oligonucleotides (PCR-ASO) in 50 individuals. The results obtained with the two methods...

  20. A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences.

    Science.gov (United States)

    Cavalcante, Gustavo Henrique O; de Araújo, Josélio M G; Fernandes, José Veríssimo; Lanza, Daniel C F

    2018-05-01

    HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Partial cytochrome b sequences for six Hymenoptera of the eastern United States.

    Science.gov (United States)

    Collins, A M; Gardner, L M

    2001-01-01

    Mitochondrial DNA (mtDNA) haplotypes have been commonly used to determine honeybee subspecies relationships. To see if these markers would also be useful for comparisons of other Hymenoptera, we collected workers of six local species: Vespa crabro, the European hornet; Bombus impatiens, a bumblebee; Vespula germanica, the German yellow jacket; Polistes fuscatus, a paper wasp; Halictus ligatus, an alkali bee; and an unspecified Megachile, a leafcutting bee. MtDNA was isolated and digested with six endonucleases (AvaI, BglII, EcoRI, HindIII, HinfI, XbaI). The digested DNA was electrophoresed and visualized on agarose gels with comparison to a standard fragment marker and similarly treated honeybee mtDNA. The fragments obtained were also purified and sequenced. Phylogenetic relationships between six wasp and bee species, Apis mellifera, and several other similar aculeate Hymenoptera were determined. Newly defined DNA sequences were posted to GenBank (AF281169-AF281174).

  2. Studies on the Interaction between Zinc-Hydroxybenzoite Complex and Genomic DNA

    Directory of Open Access Journals (Sweden)

    Hacali Necefoglu

    2006-04-01

    Full Text Available Zinc-Hydroxybenzoite ([Zn (H206] (p-HO-C6H4COO22H20 complex which wassynthesized and characterized by instrumental methods and the DNA samples which hadbeen isolated from cattle were allowed to interact at 37 oC for different time periods. Theinteraction of genomic DNA with this complex has been followed by agarose gelelectrophoresis at 50 V for 2 h. When DNA samples were allowed to interact with this metalcomplex, it was found that band intensities changed with the concentrations of the complex.In the result of interaction between this complex and genomic DNA samples, it wasdetermined that the intensities of bands were changed at the different concentrations of thecomplex. The brightness of the bands was increased and mobility of the bands wasdecreased, indicating the occurrence of increased covalent binding of the metal complexwith DNA. In this study it was concluded that the damage effect of ascorbate was reducedby Zinc-Hydroxybenzoite.

  3. Sensors and Instrumentation towards early detection of osteoporosis

    KAUST Repository

    Afsarimanesh, Nasrin

    2016-07-27

    A label-free non-invasive sensing system for detection of C-terminal telopeptide of type-I collagen (CTX-I) has been developed in order to detect bone loss at an early stage, by Electrochemical Impedance Spectroscopy (EIS). A planar interdigital sensor was functionalized by immobilizing streptavidin agarose on the sensing area of the sensor to introduce selectivity for the antigen-antibody solution. Different concentrations of CTX-I were tested using the functionalized sensing surface to capture the target molecule. Preliminary results are provided in the paper assisted with the respective equivalent circuit of the working technology on the sensor using Complex Non-linear Least Square (CNLS). The results are encouraging and will be used to develop a complete system for commercialization complementing the existing systems. © 2016 IEEE.

  4. Nano-Electrochemistry and Nano-Electrografting with an Original Combined AFM-SECM

    Directory of Open Access Journals (Sweden)

    Ammar Ben Brahim

    2013-05-01

    Full Text Available This study demonstrates the advantages of the combination between atomic force microscopy and scanning electrochemical microscopy. The combined technique can perform nano-electrochemical measurements onto agarose surface and nano-electrografting of non-conducting polymers onto conducting surfaces. This work was achieved by manufacturing an original Atomic Force Microscopy-Scanning ElectroChemical Microscopy (AFM-SECM electrode. The capabilities of the AFM-SECM-electrode were tested with the nano-electrografting of vinylic monomers initiated by aryl diazonium salts. Nano-electrochemical and technical processes were thoroughly described, so as to allow experiments reproducing. A plausible explanation of chemical and electrochemical mechanisms, leading to the nano-grafting process, was reported. This combined technique represents the first step towards improved nano-processes for the nano-electrografting.

  5. Recruitment of dental pulp cells by dentine and pulp extracellular matrix components.

    Science.gov (United States)

    Smith, J G; Smith, A J; Shelton, R M; Cooper, P R

    2012-11-01

    The present study aimed to determine whether dentine tissue and preparations of extracellular matrix (ECM) from pulp (pECM) and dentine (dECM), and breakdown products, influenced pulp cell migration. Chemotaxis transwell and agarose spot assays demonstrated that both dentine and pulp ECM molecules acted as chemoattractants for primary pulp cells. Chemoattractant activities of dECM and pECM were enhanced when subjected to acid and enzymatic breakdown, respectively. This enhanced activity following physiologically relevant breakdown may be pertinent to the disease environment. Pulp cell migration in response to dental ECMs was dependent on an active rho pathway. Recruited cells exhibited increased stem cell marker expression indicating that dental ECMs and their breakdown products selectively attract progenitor cells that contribute to repair processes. In conclusion, combined these results indicate that ECM molecules contribute to cell recruitment necessary for regeneration of the dentine-pulp complex after injury. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Effect of the caffeine on treated and non-treated plasmid DNA with stannic chloride; Efeito da cafeina em DNA plasmidial tratado e nao tratado com cloreto estanoso

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Silvana Ramos F. [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]|[Universidade Federal Fluminense, Niteroi, RJ (Brazil). Faculdade de Ciencias Medicas. Curso de Pos-graduacao em Patologia Experimental; Mattos, Jose C.P. de; Dantas, Flavio; Araujo, Adriano Caldeira de; Bernardo-Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]. E-mail: bernardo@uerj.br

    2000-07-01

    Caffeine, a methilxantine drug is a component of coffee, tea, stimulants and other drinks. Caffeine inhibits phosphodiesterase leading to intracellular accumulation of cyclic AMP, blocks adenosine receptors, and increases the release of Ca{sup 2+}. We have studied the possible effect of caffeine in DNA plasmid treated or not with stannous chloride (SnCl{sub 2}). Previous evaluations of the effect of caffeine on the labeling of red blood cells and plasma proteins with technetium-99m have showed a decrease of % ATI in the insoluble fraction of plasma proteins. Samples of DNA were treated with SnCl{sub 2} (0 and 200{mu}g/ml) in 0.8% agarose. SnCl{sub 2} has induced break on DNA and caffeine has not showed effect on the DNA. This indicates that caffeine does not eliminate the oxidant action of SnCl{sub 2} and does not promote break in isolated DNA plasmid. (author)

  7. The potential of vitamin K3 as an anticancer agent against breast cancer that acts via the mitochondria-related apoptotic pathway.

    Science.gov (United States)

    Akiyoshi, Takeshi; Matzno, Sumio; Sakai, Mika; Okamura, Noboru; Matsuyama, Kenji

    2009-12-01

    We tried to clarify the cytotoxic mechanism of VK(3) using the breast cancer cell line MCF-7. Cytotoxicity was measured via intracellular esterase activity. DNA fragmentation was assessed by agarose gel electrophoresis. JC-1 staining was applied to measure mitochondrial dysfunction. Caspase activation and reactive oxidative species (ROS) generation were also measured. VK(3) exhibited cytotoxicity that caused DNA fragmentation in MCF-7 cells with an IC(50) of 14.2 microM. JC-1 staining revealed that VK(3) caused mitochondrial dysfunction including a disappearance of mitochondrial membrane potential. Additional investigation showed that the mitochondrial damage was induced by the generation of ROS and the subsequent activation of caspase-7 and -9. Our findings demonstrate that VK(3)-induced apoptosis is selectively initiated by the mitochondria-related pathway and might be useful in breast cancer chemotherapy.

  8. The guanidinium group as a key part of water-soluble polymer carriers for siRNA complexation and protection against degradation.

    Science.gov (United States)

    Tabujew, Ilja; Freidel, Christoph; Krieg, Bettina; Helm, Mark; Koynov, Kaloian; Müllen, Klaus; Peneva, Kalina

    2014-07-01

    Here, the preparation of a novel block copolymer consisting of a statistical copolymer N-(2-hydroxypropyl) methacrylamide-s-N-(3-aminopropyl) methacrylamide and a short terminal 3-guanidinopropyl methacrylamide block is reported. This polymer structure forms neutral but water-soluble nanosized complexes with siRNA. The siRNA block copolymer complexes are first analyzed using agarose gel electrophoresis and their size is determined with fluorescence correlation spectroscopy. The protective properties of the polymer against RNA degradation are investigated by treating the siRNA block copolymer complexes with RNase V1. Heparin competition assays confirm the efficient release of the cargo in vitro. In addition, the utilization of microscale thermophoresis is demonstrated for the determination of the binding strength between a fluorescently labeled polyanion and a polymer molecule. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

    Science.gov (United States)

    van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

    1998-06-01

    DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

  10. Mapping replication origins in yeast chromosomes.

    Science.gov (United States)

    Brewer, B J; Fangman, W L

    1991-07-01

    The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.

  11. Studies on a microbially derived, high molecular weight inhibitor of plasma cholesteryl ester transfer protein

    International Nuclear Information System (INIS)

    Marschke, C.K.; McGee, J.E.; Melchior, G.W.; Castle, C.K.

    1989-01-01

    The authors have isolated an organism which accumulates an inhibitor of Cholesteryl Ester Transfer Protein (CETP). Purification of 100,000-fold was achieved by ammonium sulfate precipitation followed by Hydroxyl Apatite, Agarose AO.5, and Mono Q (Pharmacia) chromatographies. The use of 14 C-labelled protein molecular weight standards followed by SDS-PAGE revealed some proteolytic activity. However, inhibition of the proteases did not affect the inhibitor potency. The inhibitor has an estimated molecular weight of 40 Kd and appears to exist as two forms. One form was eluted from a Mono Q column by 100 mM NaCl while the other was not bound. Our evidence indicated that the bound form was progressively denatured, or proteolyzed, during storage of the fermentation beer, to the unbound form. Importantly though this molecular change did not affect either inhibitory activity or the apparent molecular weight

  12. Serial femtosecond X-ray diffraction of enveloped virus microcrystals

    Directory of Open Access Journals (Sweden)

    Robert M. Lawrence

    2015-07-01

    Full Text Available Serial femtosecond crystallography (SFX using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

  13. Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column.

    Science.gov (United States)

    Wu, Zhihua; Li, Kun; Zhan, Shaode; Tong, Ping; Li, Xin; Yang, Anshu; Chen, Hongbing

    2017-09-14

    Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits' antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.

  14. Cationization of kappa- and iota-carrageenan--Characterization and properties of amphoteric polysaccharides.

    Science.gov (United States)

    Barahona, Tamara; Prado, Héctor J; Bonelli, Pablo R; Cukierman, Ana L; Fissore, Eliana L; Gerschenson, Lia N; Matulewicz, María C

    2015-08-01

    Commercial kappa- and iota carrageenans were cationized with 3-chloro-2-hydroxypropyltrimethylammonium chloride in aqueous sodium hydroxide solution. For kappa-carrageenan three derivatives with different degrees of substitution were obtained. Native and amphoteric kappa-carrageenans were characterized by NMR and infrared spectroscopy, scanning electron and atomic force microscopy; methanolysis products were studied by electrospray ionization mass spectrometry. Young moduli and the strain at break of films, differential scanning calorimetry, rheological and flocculation behavior were also evaluated; the native and the amphoteric derivatives showed different and interesting properties. Cationization of iota-carrageenan was more difficult, indicating as it was previously observed for agarose, that substitution starts preferentially on the 2-position of 3,6-anhydrogalactose residues; in iota-carrageenan this latter unit is sulfated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Synthesis and AFM visualization of DNA nanostructures

    International Nuclear Information System (INIS)

    Mizuno, Rika; Haruta, Hirotaka; Morii, Takashi; Okada, Takao; Asakawa, Takeshi; Hayashi, Kenshi

    2004-01-01

    We propose a novel bottom-up approach for the fabrication of various desired nanostructures, based on self-assembly of oligonucleotides governed by Watson-Crick base pairing. Using this approach, we designed Y-shaped, closed Y-shaped, H-shaped, and hexagonal structures with oligonucleotides. These structures were autonomously fabricated simply by mixing equimolar solutions of oligonucleotides and performing hybridization. After synthesis of the nanostructures, we confirmed their validity by agarose gel electrophoresis and atomic force microscope (AFM) visualization. We detected bands of the desired molecular sizes in the gel electrophoresis and observed the desired structures by AFM analysis. We concluded that the synthesized structures were consistent with our intended design and that AFM visualization is a very useful tool for the observation of nanostructures

  16. High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase.

    Science.gov (United States)

    Kim, P; Yoon, S H; Roh, H J; Choi, J H

    2001-01-01

    An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.

  17. Ag nanoparticles agargel nanocomposites for SERS detection of cultural heritage interest pigments

    Science.gov (United States)

    Amato, F.; Micciche', C.; Cannas, M.; Gelardi, F. M.; Pignataro, B.; Li Vigni, M.; Agnello, S.

    2018-02-01

    Agarose gel (agargel) composites with commercial and laboratory made silver nanoparticles were prepared by a wet solution method at room temperature. The gel composites were used for pigment extraction and detection by Raman spectroscopy. Red (alizarin) and violet (crystal violet) pigments deposited on paper were extracted by the composites and were investigated by micro-Raman spectroscopy. Evaluation was carried out of the surface-enhanced Raman spectroscopy (SERS) effect induced by the silver nanoparticles embedded in the gel. A kinetic approach as a function of time was used to determine the efficiency of pigments extraction by composites deposition. A non-invasive extraction process of few minutes is demonstrated. This process induces active SERS for both used pigments. The reported results show the full exploitability of agargel silver nanoparticle composites for the extraction of pigments from paper based artworks.

  18. Hyperbaric oxygen sensitizes anoxic Pseudomonas aeruginosa biofilm to ciprofloxacin

    DEFF Research Database (Denmark)

    Kolpen, Mette; Lerche, Christian J; Kragh, Kasper Nørskov

    2017-01-01

    Chronic Pseudomonas aeruginosa lung infection is characterized by the presence of endobronchial antibiotic-tolerant biofilm subject to strong oxygen (O2) depletion due to the activity of surrounding polymorphonuclear leukocytes. The exact mechanisms affecting the antibiotic susceptibility...... metabolism activity and the endogenous formation of reactive O2 radicals (ROS). In this study we aimed to apply hyperbaric oxygen treatment (HBOT) in order to sensitize anoxic P. aeruginosa agarose-biofilms established to mimic situations with intense O2 consumption by the host response in the cystic...... fibrosis (CF) lung. Application of HBOT resulted in enhanced bactericidal activity of ciprofloxacin at clinically relevant durations and was accompanied by indications of restored aerobic respiration, involvement of endogenous lethal oxidative stress and increased bacterial growth. The findings highlight...

  19. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    Science.gov (United States)

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels. When forced through a maze of holes, the molecule unravels, forming a long chain, slithering through the field of pores in a process colloquially coined "reputation." As a result, the smaller molecules travel farther through the gel when compared to molecules of larger molecular weight. This highly effective "molecular sieve" provides consistent data and allows scientists to compare similar sequences of DNA base pairs in a routine fashion.2 When performed at the high school level, gel electrophoresis provides students the opportunity to learn about a contemporary lab technique of great scientific relevance. Doing real science certainly excites students and motivates them to learn more.

  20. Generation of multicellular tumor spheroids by the hanging-drop method.

    Science.gov (United States)

    Timmins, Nicholas E; Nielsen, Lars K

    2007-01-01

    Owing to their in vivo-like characteristics, three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are gaining increasing popularity as an in vitro model of tumors. A straightforward and simple approach to the cultivation of these MCTS is the hanging-drop method. Cells are suspended in droplets of medium, where they develop into coherent 3D aggregates and are readily accessed for analysis. In addition to being simple, the method eliminates surface interactions with an underlying substratum (e.g., polystyrene plastic or agarose), requires only a low number of starting cells, and is highly reproducible. This method has also been applied to the co-cultivation of mixed cell populations, including the co-cultivation of endothelial cells and tumor cells as a model of early tumor angiogenesis.

  1. Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy.

    Science.gov (United States)

    Schmied, Christopher; Tomancak, Pavel

    2016-01-01

    Light sheet fluorescent microscopy (LSFM), and in particular its most widespread flavor Selective Plane Illumination Microscopy (SPIM), promises to provide unprecedented insights into developmental dynamics of entire living systems. By combining minimal photo-damage with high imaging speed and sample mounting tailored toward the needs of the specimen, it enables in toto imaging of embryogenesis with high spatial and temporal resolution. Drosophila embryos are particularly well suited for SPIM imaging because the volume of the embryo does not change from the single cell embryo to the hatching larva. SPIM microscopes can therefore image Drosophila embryos embedded in rigid media, such as agarose, from multiple angles every few minutes from the blastoderm stage until hatching. Here, we describe sample mounting strategies to achieve such a recording. We also provide detailed protocols to realize multiview, long-term, time-lapse recording of Drosophila embryos expressing fluorescent markers on the commercially available Zeiss Lightsheet Z.1 microscope and the OpenSPIM.

  2. Effects of Agar Gel Strength and Fat on Oral Breakdown, Volatile Release, and Sensory Perception Using in Vivo and in Vitro Systems.

    Science.gov (United States)

    Frank, Damian; Eyres, Graham T; Piyasiri, Udayasika; Cochet-Broch, Maeva; Delahunty, Conor M; Lundin, Leif; Appelqvist, Ingrid M

    2015-10-21

    The density and composition of a food matrix affect the rates of oral breakdown and in-mouth flavor release as well as the overall sensory experience. Agar gels of increasing concentration (1.0, 1.7, 2.9, and 5% agarose) with and without added fat (0, 2, 5, and 10%) were spiked with seven aroma volatiles. Differences in oral processing and sensory perception were systematically measured by a trained panel using a discrete interval time intensity method. Volatile release was measured in vivo and in vitro by proton transfer reaction mass spectrometry. Greater oral processing was required as agar gel strength increased, and the intensity of flavor-related sensory attributes decreased. Volatile release was inversely related to gel strength, showing that physicochemical phenomena were the main mechanisms underlying the perceived sensory changes. Fat addition reduced the amount of oral processing and had differential effects on release, depending on the fat solubility or lipophilicity of the volatiles.

  3. Antimicrobial activity and safety evaluation of peptides isolated from the hemoglobin of chickens.

    Science.gov (United States)

    Hu, Fengjiao; Wu, Qiaoxing; Song, Shuang; She, Ruiping; Zhao, Yue; Yang, Yifei; Zhang, Meikun; Du, Fang; Soomro, Majid Hussain; Shi, Ruihan

    2016-12-05

    Hemoglobin is a rich source of biological peptides. As a byproduct and even wastewater of poultry-slaughtering facilities, chicken blood is one of the most abundant source of hemoglobin. In this study, the chicken hemoglobin antimicrobial peptides (CHAP) were isolated and the antimicrobial and bactericidal activities were tested by the agarose diffusion assay, minimum inhibitory concentration (MIC) analysis, minimal bactericidal concentration (MBC) analysis, and time-dependent inhibitory and bactericidal assays. The results demonstrated that CHAP had potent and rapid antimicrobial activity against 19 bacterial strains, including 9 multidrug-resistant bacterial strains. Bacterial biofilm and NaCl permeability assays, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were further performed to detect the mechanism of its antimicrobial effect. Additionally, CHAP showed low hemolytic activity, embryo toxicity, and high stability in different temperatures and animal plasma. CHAP may have great potential for expanding production and development value in animal medication, the breeding industry and environment protection.

  4. Radiation dose response of strand breaks in SINPV-DNA

    International Nuclear Information System (INIS)

    Zhang Chunxiang; Luo Daling; Li Mianfeng; Liu Xiaowei; Zeng Rong; Wang Xunzhang

    1995-01-01

    The Spodoplera litura Nuclear Polyhedrosis Viruses (SINPV) is a kind of insectile virus with a simple structure, in which a double helix DNA is encapsulated in a protein coat and there is no function of enzymatic repair. The SINPV samples in dry powdered form held in sealed plastic tube were irradiated by 1-100 kGy gamma rays. The single strand breaks (SSB) and double strand breaks (DSB) induced in SINPV after irradiation were measured by neutral and alkaline agarose gel electrophoresis. A dose-response function combining the responses of one-hit and two-hit events was used to describe the SSB and DSB dose-response curves. It is shown that the SSB are one-hit events and the DSB are the combination of both one-hit, and two-hit events, and two-hit events are predominant in the DSB process

  5. Apoptosis - Triggering Effects: UVB-irradiation and Saccharomyces cerevisiae.

    Science.gov (United States)

    Behzadi, Payam; Behzadi, Elham

    2012-12-01

    The pathogenic disturbance of Saccharomyces cerevisiae is known as a rare but invasive nosocomial fungal infection. This survey is focused on the evaluation of apoptosis-triggering effects of UVB-irradiation in Saccharomyces cerevisiae. The well-growth colonies of Saccharomyces cerevisiae on Sabouraud Dextrose Agar (SDA) were irradiated within an interval of 10 minutes by UVB-light (302 nm). Subsequently, the harvested DNA molecules of control and UV-exposed yeast colonies were run through the 1% agarose gel electrophoresis comprising the luminescent dye of ethidium bromide. No unusual patterns including DNA laddering bands or smears were detected. The applied procedure for UV exposure was not effective for inducing apoptosis in Saccharomyces cerevisiae. So, it needs another UV-radiation protocol for inducing apoptosis phenomenon in Saccharomyces cerevisiae.

  6. Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.

    Science.gov (United States)

    Chee Wei, T; Nurul Wahida, A G; Shaharum, S

    2014-12-01

    Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

  7. A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

    Science.gov (United States)

    Govan, V A; Brözel, V; Allsopp, M H; Davison, S

    1998-05-01

    Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

  8. A noda-like virus isolated from the sweetpotato pest spodoptera eridania (Cramer) (Lep.; noctuidae)

    Science.gov (United States)

    Zeddam; Rodriguez; Ravallec; Lagnaoui

    1999-11-01

    A small isometric virus has been isolated from larvae of the sweetpotato pest Spodoptera eridania (Cramer) collected near Pariacoto, Ancash province, Peru. It is designated the Pariacoto virus (PaV). In addition to its high pathogenicity on its natural host Spodoptera eridania, PaV was found to replicate in Spodoptera ochrea (Hampson) larvae but not in Spodoptera frugiperda (Smith) larvae. The size of the viral particle was estimated to be about 30 nm in diameter. Polyacrylamide gel electrophoresis showed a protein of approximately 40.5 kDa. After agarose gel electrophoresis, the viral genome appeared to be bipartite RNA. Gel immunodiffusion tests showed no serological relationship between PaV and Nodamura virus, the type species for insect nodaviruses. Electron microscopy confirmed that viral replication occurs in the cytoplasm. These properties are similar to those of other members of family Nodaviridae, to which the virus is currently assigned. Copyright 1999 Academic Press.

  9. First molecular detection of Chronic Bee Paralysis Virus (CBPV in Iran

    Directory of Open Access Journals (Sweden)

    Modirrousta, H.

    2015-12-01

    Full Text Available Among the viruses infecting honey bees, chronic bee paralysis virus (CBPV is known to induce significant losses in honey bee colonies. CBPV is an unclassified polymorphic single stranded RNA virus. Using RT-PCR, the virus infections in honey bees can be detected in a rapid and accurate manner. Bee samples were collected from 23 provinces of Iran, between July-September 2011 and 2012. A total of 160 apiaries were sampled and submitted for virus screening. RNA extraction and RT-PCR were performed with QIAGEN kits. The primers lead to a fragment of 315 bp. The PCR products were electrophoresed in a 1.2 % agarose gel. Following the RT-PCR reaction with the specific primers, out of the 160 apiaries examined, 12 (7.5 % were infected with CBPV. This is the first study of CBPV detection in Iranian apiaries. We identified CBPV in the collected samples from different geographic regions of Iran.

  10. Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

    2014-11-01

    Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

    International Nuclear Information System (INIS)

    Zhao Xiaoli; Li Zhaoyang; Liu Wenguang; Lam, Wingmoon; Sun Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

    2011-01-01

    Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R 8 -CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R 8 -CSs were characterized using FTIR and 1 H NMR. Agarose gel electrophoresis assay showed that R 8 -CS could efficiently condense the DNA. The particle size of R 8 -CS/DNA complexes were determined to be around 100–200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R 8 -CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

  12. Understanding the Effect of Carbonate Ion on Cisplatin Binding to DNA

    Science.gov (United States)

    Todd, Ryan C.; Lovejoy, Katherine S.; Lippard, Stephen J.

    2008-01-01

    The role of carbonate in the binding of cis-diamminedichloroplatinum(II) to DNA was investigated in order to understand the potential involvement of carbonato-cisplatin species in the mechanism of action of platinum anticancer agents. Cisplatin was allowed to react with both double- and single-stranded DNA in carbonate, phosphate, and HEPES buffers, and the products were analyzed by agarose gel electrophoresis and enzymatic digestion/mass spectrometry, respectively. The data from these experiments demonstrate (1) that carbonate, like other biological nucleophiles, forms relatively inert complexes with platinum that inactivate cisplatin, and (2) that the major cisplatin-DNA adduct formed is a bifunctional cross-link. These results are in accord with previous studies of cisplatin-DNA binding and reveal that the presence of carbonate has no consequence on the nature of the resulting adducts. PMID:17465550

  13. Gender identification of Grasshopper Sparrows comparing behavioral, morphological, and molecular techniques

    Science.gov (United States)

    Ammer, F.K.; Wood, P.B.; McPherson, R.J.

    2008-01-01

    Correct gender identification in monomorphic species is often difficult especially if males and females do not display obvious behavioral and breeding differences. We compared gender specific morphology and behavior with recently developed DNA techniques for gender identification in the monomorphic Grasshopper Sparrow (Ammodramus savannarum). Gender was ascertained with DNA in 213 individuals using the 2550F/2718R primer set and 3% agarose gel electrophoresis. Field observations using behavior and breeding characteristics to identify gender matched DNA analyses with 100% accuracy for adult males and females. Gender was identified with DNA for all captured juveniles that did not display gender specific traits or behaviors in the field. The molecular techniques used offered a high level of accuracy and may be useful in studies of dispersal mechanisms and winter assemblage composition in monomorphic species.

  14. A flexible micro biofuel cell utilizing hydrogel containing ascorbic acid

    Science.gov (United States)

    Goto, Hideaki; Fukushi, Yudai; Nishioka, Yasushiro

    2014-11-01

    This paper reports on a biofuel cell with a dimension of 13×24 mm2 fabricated on a flexible polyimide substrate. I its porous carbon-coated platinum (Pt) electrodes of 3 mm in width and 10 mm in length were fabricated using photolithography and screen printing techniques. Porous carbon was deposited by screen printing of carbon black ink on the Pt electrode surfaces in order to increase the effective electrode surface area and to absorb more enzymes on the electrode surfaces. It utilizes a solidified ascorbic acid (AA) aqueous solution in an agarose hydrogel to increase the portability. The maximum power and power density for the biofuel cell with the fuel unit containing 100 mM AA were 0.063 μW and 0.21 μW/cm2 at 0.019 V, respectively.

  15. A flexible micro biofuel cell utilizing hydrogel containing ascorbic acid

    International Nuclear Information System (INIS)

    Goto, Hideaki; Fukushi, Yudai; Nishioka, Yasushiro

    2014-01-01

    This paper reports on a biofuel cell with a dimension of 13×24 mm 2 fabricated on a flexible polyimide substrate. I its porous carbon-coated platinum (Pt) electrodes of 3 mm in width and 10 mm in length were fabricated using photolithography and screen printing techniques. Porous carbon was deposited by screen printing of carbon black ink on the Pt electrode surfaces in order to increase the effective electrode surface area and to absorb more enzymes on the electrode surfaces. It utilizes a solidified ascorbic acid (AA) aqueous solution in an agarose hydrogel to increase the portability. The maximum power and power density for the biofuel cell with the fuel unit containing 100 mM AA were 0.063 μW and 0.21 μW/cm 2 at 0.019 V, respectively

  16. How many 5S rRNA genes and pseudogenes are there in ''Aspergillus nidulans''?

    International Nuclear Information System (INIS)

    Pelczar, P.; Fiett, J.; Bartnik, E.

    1994-01-01

    We have estimated the number of 5S rRNA genes in ''Aspergillus nidulans'' using two-dimensional agarose gel electrophoresis and hybridization to appropriate probes, representing the 5'-halves, the 3'-halves of the 5S rRNA sequence and a sequence found at the 3'-end of all known. ''A. nidulans'' pseudogenes (block C). We have found 23 5S rRNA genes, 15 pseudogenes consisting of the 5'-half of the 5S rRNA sequence (of which 3 are flanked by block C) and 12 copies of block C which do not seem to be in the vicinity of 5S rRNA sequences. This number of genes is much lower than our earlier estimates, and makes our previously analyzed sample of 9 sequenced genes and 3 pseudogenes much more representative. (author). 7 refs, 1 fig

  17. Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes

    DEFF Research Database (Denmark)

    Fischer-Nielsen, A; Corcoran, G B; Poulsen, H E

    1995-01-01

    Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly...... damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 micro......M, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative...

  18. Rheological and DSC study of sol-gel transition in aqueous dispersions of industrially important polymers and colloids

    Energy Technology Data Exchange (ETDEWEB)

    Nishinari, K. [Osaka City Univ. (Japan). Dept. of Food and Nutrition

    1997-12-01

    Gelation kinetics, mechanical spectra, thermal scanning rheology (TSR), and differential scanning calorimetry (DSC) in aqueous solutions of gelling polymers and colloids such as seaweed polysaccharides (agarose, carrageenans), microbial polysaccharides (gellan, curdlan), plant polysaccharides (methylcellulose), globular proteins (casein, glycinin, {beta}-conglycinin), fibrous proteins (gelatin, fibrin), and polyvinyl alcohol, which are related to foods, cosmetics, biomedical and pharmaceutical applications, are described. Some gelation processes at a constant temperature have been treated successfully by an equation of first order kinetics or by other modified equations, and the molecular mechanism of gel formation is discussed briefly. For water-soluble polymers, the criterion of the gel or sol based on the frequency dependence of storage and loss moduli gives valuable informations. TSR and DSC are complementary, and the combination of these methods has been proved to be useful. (orig.) 81 refs.

  19. Catalytic effects by thioltransferase on the transfer of methylmercury and p-mercuribenzoate from macromolecules to low molecular weight thiol compounds

    Energy Technology Data Exchange (ETDEWEB)

    Eriksson, S.; Svenson, A.

    1978-01-01

    Thiol agarose and glyceraldehyde-3-phosphate dehydrogenase were blocked with methylmercury or p-mercuribenzoate. The exchange of mercurials between the thiol-containing polymers and glutathione or dithioerythritol was investigated. The activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited by blocking thiol-groups with the mercury compounds. Inhibition was reversible when a short period of inactivation was used. Inactivation for longer periods resulted in reduced regain of enzyme activity. The activity was in part regained when either of the 2 thiol compounds was added. Thioltransferase, known to catalyze thiol-disulfide exchange reactions, increased the regain of glyceraldehyde-3-phosphate dehydrogenase activity to nearly the original value. Here, thioltransferase is proposed to catalyze the transfer of organomercurial from one thiol complex to another. Some consequences of the observations in vivo are discussed.

  20. Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa.

    Science.gov (United States)

    Matsumoto, E

    1991-02-01

    Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.