Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR.

PubMed

Leng, Xuefei; Zhang, Wenhua; Wang, Chunming; Cui, Liang; Yang, Chaoyong James

2010-11-07

An agarose droplet method was developed for highly parallel and efficient single molecule emulsion PCR. The method capitalizes on the unique thermoresponsive sol-gel switching property of agarose for highly efficient DNA amplification and amplicon trapping. Uniform agarose solution droplets generated via a microfluidic chip serve as robust and inert nanolitre PCR reactors for single copy DNA molecule amplification. After PCR, agarose droplets are gelated to form agarose beads, trapping all amplicons in each reactor to maintain the monoclonality of each droplet. This method does not require cocapsulation of primer labeled microbeads, allows high throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.

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.

PubMed

Pedersen, T V; Olsen, D R; Skretting, A

1997-08-01

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 mm2 h-1, at the cost of significantly lower Rl sensitivity. The addition of benzoic acid to the latter gel did not increase the Rl sensitivity.

Towards more realistic in vitro release measurement techniques for biodegradable microparticles.

PubMed

Klose, D; Azaroual, N; Siepmann, F; Vermeersch, G; Siepmann, J

2009-03-01

To better understand the importance of the environmental conditions for drug release from biodegradable microparticles allowing for the development of more appropriate in vitro release measurement techniques. Propranolol HCl diffusion in various agarose gels was characterized by NMR and UV analysis. Fick's law was used to theoretically predict the mass transport kinetics. Drug release from PLGA-based microparticles in such agarose gels was compared to that measured in agitated bulk fluids ("standard" method). NMR analysis revealed that the drug diffusivity was almost independent of the hydrogel concentration, despite of the significant differences in the systems' mechanical properties. This is due to the small size of the drug molecules/ions with respect to the hydrogel mesh size. Interestingly, the theoretically predicted drug concentration-distance-profiles could be confirmed by independent experiments. Most important from a practical point of view, significant differences in the release rates from the same batch of PLGA-based microparticles into a well agitated bulk fluid versus a semi-solid agarose gel were observed. Great care must be taken when defining the in vitro conditions for drug release measurements from biodegradable microparticles. The obtained new insight can help facilitating the development of more appropriate in vitro release testing procedures.

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

PubMed

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.

A Plasmid Containing the Human Metallothionein II Gene Can Function as an Antibody-assisted Electrophoretic Biosensor for Heavy Metals

DTIC Science & Technology

2015-01-16

this agarose gel-based method might be useful in heavy metal bioavailability testing of aqueous samples from a variety of sources ( water treatment ...key biological proteins that protect cells from heavy metal poisoning. The gel-based method may be utilized in water treatment facilities or on...currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 20 Feb 2015 2. REPORT TYPE

Visualization of yeast chromosomal DNA

NASA Technical Reports Server (NTRS)

Lubega, Seth

1990-01-01

The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

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

PubMed

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.

Wheat gliadin: digital imaging and database construction using a 4-band reference system of agarose isoelectric focusing patterns.

PubMed

Black, J A; Waggamon, K A

1992-01-01

An isoelectric focusing method using thin-layer agarose gel has been developed for wheat gliadin. Using flat-bed units with a third electrode, up to 72 samples per gel may be analyzed. Advantages over traditional acid polyacrylamide gel electrophoresis methodology include: faster run times, nontoxic media, and greater sample capacity. The method is suitable for fingerprinting or purity testing of wheat varieties. Using digital images captured by a flat-bed scanner, a 4-band reference system using isoelectric points was devised. Software enables separated bands to be assigned pI values based upon reference tracks. Precision of assigned isoelectric points is shown to be on the order of 0.02 pH units. Captured images may be stored in a computer database and compared to unknown patterns to enable an identification. Parameters for a match with a stored pattern may be adjusted for pI interval required for a match, and number of best matches.

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

PubMed

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.

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

NASA Astrophysics Data System (ADS)

Marrale, Maurizio; Collura, Giorgio; Gallo, Salvatore; Nici, Stefania; Tranchina, Luigi; Abbate, Boris Federico; Marineo, Sandra; Caracappa, Santo; d'Errico, Francesco

2017-04-01

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.

A rapid method to visualize von willebrand factor multimers by using agarose gel electrophoresis, immunolocalization and luminographic detection.

PubMed

Krizek, D R; Rick, M E

2000-03-15

A highly sensitive and rapid clinical method for the visualization of the multimeric structure of von Willebrand Factor in plasma and platelets is described. The method utilizes submerged horizontal agarose gel electrophoresis, followed by transfer of the von Willebrand Factor onto a polyvinylidine fluoride membrane, and immunolocalization and luminographic visualization of the von Willebrand Factor multimeric pattern. This method distinguishes type 1 from types 2A and 2B von Willebrand disease, allowing timely evaluation and classification of von Willebrand Factor in patient plasma. It also allows visualization of the unusually high molecular weight multimers present in platelets. There are several major advantages to this method including rapid processing, simplicity of gel preparation, high sensitivity to low concentrations of von Willebrand Factor, and elimination of radioactivity.

Agarose template for the fabrication of macroporous metal oxide structures.

PubMed

Zhou, Jingfang; Zhou, Meifang; Caruso, Rachel A

2006-03-28

Agarose gels have been applied as templates for the formation of macroporous metal oxide structures. The preparation of the agarose template is extremely simple, and with variation of the agarose content, control over morphology is demonstrated: The average pore size decreases from 180 to 55 nm and the surface area increases from 238 to 271 m2 g(-1) with increasing agarose content in the gel. The gelling temperature was also found to influence the final template morphology. Conducting sol-gel chemistry within the template structure followed by removal of the template by heating to 450 degrees C gives porous inorganic oxides. The technique has been demonstrated for the oxides of titanium, zirconium, niobium, and tin. The final morphology of the metal oxide is homogeneous and results from a coating of the agarose structure. The pore diameter decreased and the specific surface area of the titanium dioxide materials increased from 28 to 66 m2 g(-1) as the agarose content in the template is increased from 0.5 to 5.0 wt%. The overall pore size and surface area are lower than the original gel due to shrinkage occurring with the sol-gel process, as well as crystallization and a loss of microporosity in the final material.

Anomalous diffusion of poly(ethylene oxide) in agarose gels.

PubMed

Brenner, Tom; Matsukawa, Shingo

2016-11-01

We report on the effect of probe size and diffusion time of poly(ethylene) oxide in agarose gels. Time-dependence of the diffusion coefficient, reflecting anomalous diffusion, was observed for poly(ethylene) oxide chains with hydrodynamic radii exceeding about 20nm at an agarose concentration of 2%. The main conclusion is that the pore distribution includes pores that are only several nm across, in agreement with scattering reports in the literature. Interpretation of the diffusion coefficient dependence on the probe size based on a model of entangled rigid rods yielded a rod length of 72nm. Copyright © 2016. Published by Elsevier B.V.

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

PubMed

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.

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

PubMed

Li, Xingrui; Zhang, Dongfeng; Zhang, Huimin; Guan, Zhichao; Song, Yanling; Liu, Ruochen; Zhu, Zhi; Yang, Chaoyong

2018-02-20

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

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

NASA Astrophysics Data System (ADS)

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.

Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

PubMed

Hegedüs, Eva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

2009-09-01

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

Detection of foodborne pathogens using microarray technology

USDA-ARS?s Scientific Manuscript database

Assays based on the polymerase chain reaction (PCR) are now accepted methods for rapidly confirming the presence or absence of specific pathogens in foods and other types of samples. Conventional PCR requires the use of agarose gel electrophoresis to detect the PCR product; whereas, real-time PCR c...

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

PubMed

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 <5% for all protein fractions. No significant difference was found between warm-blooded and heavy 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.

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

1987-09-04

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

Study of kinetic desorption rate constant in fish muscle and agarose gel model using solid phase microextraction coupled with liquid chromatography with tandem mass spectrometry.

PubMed

Togunde, Oluranti Paul; Oakes, Ken; Servos, Mark; Pawliszyn, Janusz

2012-09-12

This study aims to use solid phase microextraction (SPME), a simple tool to investigate diffusion rate (time) constant of selected pharmaceuticals in gel and fish muscle by comparing desorption rate of diffusion of the drugs in both agarose gel prepared with phosphate-buffered saline (PBS; pH 7.4) and fish muscle. The gel concentration (agarose gel model) that could be used to simulate tissue matrix (fish muscle) for free diffusion of drugs under in vitro and in vivo conditions was determined to model mass transfer phenomena between fibre polymer coating and environmental matrix such that partition coefficients and desorption time constant (diffusion coefficient) can be determined. SPME procedure involves preloading the extraction phase (fibre) with the standards from spiked PBS for 1h via direct extraction. Subsequently, the preloaded fibre is introduced to the sample such fish or agarose gel for specified time ranging from 0.5 to 60 h. Then, fibre is removed at specified time and desorbed in 100 μL of desorption solution (acetonitrile: water 1:1) for 90 min under agitation speed of 1000 rpm. The samples extract were immediately injected to the instrument and analysed using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The limit of detection of the method in gel and fish muscle was 0.01-0.07 ng mL(-1) and 0.07-0.34 ng g(-1), respectively, while the limit quantification was 0.10-0.20 ng mL(-1) in gel samples and 0.40-0.97 ng g(-1) in fish sample. The reproducibility of the method was good (5-15% RSD). The results suggest that kinetics of desorption of the compounds in fish tissue and different viscosity of gel can be determined using desorption time constant. In this study, desorption time constant which is directly related to desorption rate (diffusion kinetics) of selected drugs from the fibre to the gel matrix is faster as the viscosity of the gel matrix reduces from 2% (w/v) to 0.8% (w/v). As the concentration of gel reduces, viscosity of the gel will be reduced therefore allowing faster diffusion which invariably affect desorption time constant. Also, desorption time constant of model drugs in the fish muscle and 0.8-0.9% (w/v) gel model are similar based on free diffusion of studied compounds. In addition, in vitro and in vivo desorption time constant comparison shows that desorption time constant in an in vivo system (live fish muscle) is generally higher than an in vitro system (dead fish muscle) except for sertraline and nordiazepam. This study demonstrates SPME as a simple investigative tool to understand kinetics of desorption in an in vivo system with a goal to measure desorption rate of pharmaceuticals in fish. Copyright © 2011 Elsevier B.V. All rights reserved.

Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armstrong, G.D.; Peppler, M.S.

1987-05-01

We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigationsmore » into the nature of the interaction and activity of PT in host tissues.« less

Bleach Gel: A Simple Agarose Gel for Analyzing RNA Quality

PubMed Central

Aranda, Patrick S.; LaJoie, Dollie M.; Jorcyk, Cheryl L.

2013-01-01

RNA-based applications requiring high quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the ‘bleach gel’ is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality. PMID:22222980

Role of Electrostatic Interactions on the Transport of Druglike Molecules in Hydrogel-Based Articular Cartilage Mimics: Implications for Drug Delivery.

PubMed

Ye, Fengbin; Baldursdottir, Stefania; Hvidt, Søren; Jensen, Henrik; Larsen, Susan W; Yaghmur, Anan; Larsen, Claus; Østergaard, Jesper

2016-03-07

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/v) chondroitin sulfate or 0.5% (w/v) hyaluronic acid were developed, and their rheological and morphological properties were characterized. UV imaging was utilized to quantify the transport properties of the following four model compounds in the agarose gel and in the developed artificial cartilage models: H-Ala-β-naphthylamide, H-Lys-Lys-β-naphthylamide, lysozyme, and α-lactalbumin. The obtained results showed that the incorporation of the polyelectrolytes chondroitin sulfate or hyaluronic acid into agarose gel induced a significant reduction in the apparent diffusivities of the cationic model compounds as compared 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 in the negatively charged hydrogels was highly dependent on the ionic strength, pointing out the importance of electrostatic interactions between the diffusant and the polyelectrolytes. Solution based affinity studies between the model compounds and the two investigated polyelectrolytes further confirmed 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 release testing having features resembling the articular cartilage.

Methods and reagents. Degraded DNA and gel tornados.

PubMed

Hengen, P N

1997-04-01

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a case of inexplicable DNA degradation and tornados seen in agarose gels. For details on how to partake in the newsgroup, see the accompanying box.

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

PubMed

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.

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials.

PubMed

Dawes, Clive C; Jewess, Philip J; Murray, Deborah A

2005-03-15

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.

Topology evolution and gelation mechanism of agarose gel.

PubMed

Xiong, Jun-Ying; Narayanan, Janaky; Liu, Xiang-Yang; Chong, Tan Kok; Chen, Shing Bor; Chung, Tai-Shung

2005-03-31

Kinetics as well as the evolution of the agarose gel topology is discussed, and the agarose gelation mechanism is identified. Aqueous high melting (HM) agarose solution (0.5% w/v) is used as the model system. It is found that the gelation process can be clearly divided into three stages: induction stage, gelation stage, and pseudoequilibrium stage. The induction stage of the gelation mechanism is identified using an advanced rheological expansion system (ARES, Rheometric Scientific). When a quench rate as large as 30 deg C/min is applied, gelation seems to occur through a nucleation and growth mechanism with a well-defined induction time (time required for the formation of the critical nuclei which enable further growth). The relationship between the induction time and the driving force which is determined by the final setting temperature follows the 3D nucleation model. A schematic representation of the three stages of the gelation mechanism is given based on turbidity and rheological measurements. Aggregation of agarose chains is promoted in the polymer-rich phase and this effect is evident from the increasing mass/length ratio of the fiber bundles upon gelation. Continuously increasing pore size during gelation may be attributed to the coagulation of the local polymer-rich phase in order to achieve the global minimum of the free energy of the gelling system. The gel pore size determined using turbidity measurements has been verified by electrophoretic mobility measurements.

Hindered diffusion of high molecular weight compounds in brain extracellular microenvironment measured with integrative optical imaging.

PubMed

Nicholson, C; Tao, L

1993-12-01

This paper describes the theory of an integrative optical imaging system and its application to the analysis of the diffusion of 3-, 10-, 40-, and 70-kDa fluorescent dextran molecules in agarose gel and brain extracellular microenvironment. The method uses a precisely defined source of fluorescent molecules pressure ejected from a micropipette, and a detailed theory of the intensity contributions from out-of-focus molecules in a three-dimensional medium to a two-dimensional image. Dextrans tagged with either tetramethylrhodamine or Texas Red were ejected into 0.3% agarose gel or rat cortical slices maintained in a perfused chamber at 34 degrees C and imaged using a compound epifluorescent microscope with a 10 x water-immersion objective. About 20 images were taken at 2-10-s intervals, recorded with a cooled CCD camera, then transferred to a 486 PC for quantitative analysis. The diffusion coefficient in agarose gel, D, and the apparent diffusion coefficient, D*, in brain tissue were determined by fitting an integral expression relating the measured two-dimensional image intensity to the theoretical three-dimensional dextran concentration. The measurements in dilute agarose gel provided a reference value of D and validated the method. Values of the tortuosity, lambda = (D/D*)1/2, for the 3- and 10-kDa dextrans were 1.70 and 1.63, respectively, which were consistent with previous values derived from tetramethylammonium measurements in cortex. Tortuosities for the 40- and 70-kDa dextrans had significantly larger values of 2.16 and 2.25, respectively. This suggests that the extracellular space may have local constrictions that hinder the diffusion of molecules above a critical size that lies in the range of many neurotrophic compounds.

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

NASA Astrophysics Data System (ADS)

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.

Dosimetry Evolution in Teletherapy: Polimer Gel

NASA Astrophysics Data System (ADS)

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

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

NASA Astrophysics Data System (ADS)

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.

Differential quantification of SIgA and SC by two-directional rocket method.

PubMed Central

Kosaka, T; Asahina, T; Kobayashi, N

1980-01-01

The two-directional rocket method, a newly modified method for quantitative immunoelectrophoresis, was used as the assay for separating SC and SIgA, which have identical antigenicity but differ in mobility. This method proved to be sufficiently simple and sensitive to enable simultaneous assay of SC and SIgA in saliva. The method employs electrophoresis into antibody-containing agarose/agar gel in the presence of heparin-Ca EDTA. The height of the precipitation peaks formed in two directions is proportional to the concentration of the antigens. Concomitant use of agarose which has little electroendosmosis and agar which has high electroendosmosis facilitated cathodic migration of SIgA. Transfer of SC from beta-region to alpha 1-region without influencing the mobilities of SIgA, albumin or IgG was obtained by addition of heparin-Ca EDTA to agarose/agar gel. This effect of heparin-Ca EDTA is vulnerable to changes of pH of the gel, but is almost completely independent of change in composition or concentration of the gel. The function of heparin as a polyanion may be resonsible for it. Carbamylation of antibody was used to accelerate a clear-cut resolution of the cathodic rockets. This technique was found to be a method of choice for analysis of SIgA and SC in large numbers. In using this method as a screening assay for detection of primary immunodeficiency, by studying saliva samples collected from 3 month old infants on the occasion of regular check-up over a 2 year period, two cases of isolated IgA immunodeficiency and two cases of hypoglobulinaemia were discovered in 12,000 infants. Images Figure 1 Figure 7 PMID:6776036

Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

DOEpatents

Zhang, Jian-Shi; Giometti, Carol S.; Tollaksen, Sandra L.

1989-01-01

After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

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

PubMed

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.

An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.

PubMed

Lee, H G; Cowman, M K

1994-06-01

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Procedures and computer program for deriving the Ferguson plot from electrophoresis in a single pore gradient gel: application to agarose gel and a polystyrene particle.

PubMed

Tietz, D; Gombocz, E; Chrambach, A

1991-10-01

This study presents a computerized evaluation of pore gradient gel electrophoretograms to arrive at estimates for both the particle-free mobility and retardation coefficient, which is related to particle size. Agarose pore gradient gels ranging from 0.2 to 1.1% agarose were formed. Gel gradients were stabilized during their formation by a density gradient of 0-20% 5-(N-2,3-dihydroxypropylacetamido)- 2,4,6-triiodo-N,N'bis-(2,3-dihydroxypropyl)-isophthalamide (Nycodenz). Densitometry of gelled-in Bromophenol Blue showed that these pore gradients exhibited a linear central segment and were reproducible. Migration distances of polystyrene sulfate microspheres (36.5 nm radius) in agarose pore gradient gel electrophoresis were determined by time-lapse photography at several durations of electrophoresis. These migration distances were evaluated as a function of migration time as previously reported (D. Tietz, Adv. Electrophoresis 1988, 2, 109-169). Although this is not necessarily required, the mathematical approach used in this study assumed linearity of both the pore gradient and the Ferguson plot for reasons of simplicity. The data evaluation on the basis of the extended Ogston model is incorporated in a user-friendly program, GRADFIT, which is designed for personal computers (Macintosh). The results obtained are compared with (1) conventional electrophoresis using several gels of single concentration with and without Nycodenz, and (ii) a different mathematical approach for the analysis of gradient gels (Rodbard et al., Anal. Biochem. 1971, 40, 135-157). Moreover, a simple procedure for evaluating linear pore gradient gels using linear regression analysis is presented. It is concluded that the values of particle-free mobility and retardation coefficient derived from pore gradient gel electrophoresis using the different mathematical methods are statistically indistinguishable from each other. However, these values are different, albeit close, to those obtained from conventional Ferguson plots. One of the possible reasons for this relatively minor discrepancy is that the particle-free mobility changed slightly during electrophoresis, which has a different effect on electrophoresis in homogeneous gels (single time measurement) and pore gradient gels (multiple time measurements). The characterization of particles according to size and charge by pore gradient electrophoresis provides a significant operational simplification and sample economy compared to that requiring the use of several gel concentrations, although at the price of increased requirements of instrumentation.

Introduction of agarose gel as a green membrane in electromembrane extraction: An efficient procedure for the extraction of basic drugs with a wide range of polarities.

PubMed

Tabani, Hadi; Asadi, Sakine; Nojavan, Saeed; Parsa, Mitra

2017-05-12

Developing green methods for analyte extraction is one of the most important topics in the field of sample preparation. In this study, for the first time, agarose gel was used as membrane in electromembrane extraction (EME) without using any organic solvent, for the extraction of four model basic drugs (rivastigmine (RIV), verapamil (VER), amlodipine (AML), and morphine (MOR)) with a wide polarity window (log P from 0.43 to 3.7). Different variables playing vital roles in the proposed method were evaluated and optimized. As a driving force, a 25V electrical field was applied to make the analyte migrate from sample solution with pH 7.0, through the agarose gel 3% (w/v) with 5mm thickness, into an acceptor phase (AP) with pH 2.0. The best extraction efficiency was obtained with an extraction duration of 25min. With this new methodology, MOR with high polarity (log P=0.43) was efficiently extracted without using any carrier or ion pair reagents. Limits of detection (LODs) and quantification (LOQs) were in the ranges of 1.5-1.8ngmL -1 and 5.0-6.0ngmL -1 , respectively. Finally, the proposed method was successfully applied to determine concentrations of the model drugs in the wastewater sample. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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.

Development of a highly sensitive three-dimensional gel electrophoresis method for characterization of monoclonal protein heterogeneity.

PubMed

Nakano, Keiichi; Tamura, Shogo; Otuka, Kohei; Niizeki, Noriyasu; Shigemura, Masahiko; Shimizu, Chikara; Matsuno, Kazuhiko; Kobayashi, Seiichi; Moriyama, Takanori

2013-07-15

Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed Central

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

Agarose gel electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA system as a new prognostic tool for periprosthetic osteolysisin revision arthroplasty

PubMed Central

Chiva, A

2013-01-01

Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis (products of wear, bone turnover and inflammatory biomarkers), the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice. Objective. The aim of this paper is to present three new electrophoretic methods using Hyrys-Hydrasys SEBIA system that have been used for the first time in Electrophoresis Laboratory of our hospital in the analysis of joint fluid for the prevention of periprosthetic osteolysis in revision arthroplasty. Methods and results. Analytical aspects of agarose gel electrophoresis of joint fluid proteins and lipoproteins as well as SDS-agarose gel electrophoresis of joint fluid proteins, their performances and clinical value are presented. The decreased level of albumin and increased level of alpha1 and alpha2 globulins were frequent changes detected on SEBIA electropherograms and good indicator for the presence of an inflammatory reaction generated by particle debris. In addition, a slightly increase of LDL mobility could provide good information about a high oxidative stress. Moreover, the Ig G assessed by using SDS-agarose gel electrophoresis could be a potential biomarker for an immunological reaction towards orthopedic implants. Discussion. Electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA France system is a new analytical technique able to remove the most of current biomarkers disadvantages due to the determination of particular proteins (acute phase proteins, albumin, lipoproteins, and immunoglobulins) by using minimal amounts of joint fluid with minor interferences, minimal cost and rapid results. Abbreviations CTX, crosslinked C-telopeptide; IL- interleukins; Ig G, immunoglobulin G; LDL, low density lipoprotein; NTX, crosslinked N-telopeptide; PICP, procollagen I C – terminal extension peptide; SDS, sodium dodecyl sulphate PMID:24146682

Function, structure, and stability of enzymes confined in agarose gels.

PubMed

Kunkel, Jeffrey; Asuri, Prashanth

2014-01-01

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.

Southern blotting.

PubMed

Brown, T

2001-05-01

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid than the basic protocol and can result in more complete transfer. Although the ease and reliability of capillary transfer methods makes this far and away the most popular system for Southern blotting with agarose gels, it unfortunately does not work with polyacrylamide gels, whose smaller pore size impedes the transverse movement of the DNA molecules. The third alternate protocol describes an electroblotting procedure that is currently the most reliable method for transfer of DNA from a polyacrylamide gel. Dot and slot blotting are also described.

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

PubMed

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.

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

ERIC Educational Resources Information Center

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…

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

PubMed

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.

DNA electrophoresis in agarose gels: effects of field and gel concentration on the exponential dependence of reciprocal mobility on DNA length.

PubMed

Rill, Randolph L; Beheshti, Afshin; Van Winkle, David H

2002-08-01

Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation nu(L) = (beta+alpha(1+exp(-L/gamma))(-1) can be used to relate mobility to length with high accuracy. Mobility data were fit with chi(2) > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.

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

PubMed

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.

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

PubMed Central

2010-01-01

Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research. PMID:20180960

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

PubMed

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.

Technical Note: Radiological properties of tissue surrogates used in a multimodality deformable pelvic phantom for MR-guided radiotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niebuhr, Nina I., E-mail: n.niebuhr@dkfz.de; Johnen, Wibke; Güldaglar, Timur

Purpose: Phantom surrogates were developed to allow multimodal [computed tomography (CT), magnetic resonance imaging (MRI), and teletherapy] and anthropomorphic tissue simulation as well as materials and methods to construct deformable organ shapes and anthropomorphic bone models. Methods: Agarose gels of variable concentrations and loadings were investigated to simulate various soft tissue types. Oils, fats, and Vaseline were investigated as surrogates for adipose tissue and bone marrow. Anthropomorphic shapes of bone and organs were realized using 3D-printing techniques based on segmentations of patient CT-scans. All materials were characterized in dual energy CT and MRI to adapt CT numbers, electron density, effectivemore » atomic number, as well as T1- and T2-relaxation times to patient and literature values. Results: Soft tissue simulation could be achieved with agarose gels in combination with a gadolinium-based contrast agent and NaF to simulate muscle, prostate, and tumor tissues. Vegetable oils were shown to be a good representation for adipose tissue in all modalities. Inner bone was realized using a mixture of Vaseline and K{sub 2}HPO{sub 4}, resulting in both a fatty bone marrow signal in MRI and inhomogeneous areas of low and high attenuation in CT. The high attenuation of outer bone was additionally adapted by applying gypsum bandages to the 3D-printed hollow bone case with values up to 1200 HU. Deformable hollow organs were manufactured using silicone. Signal loss in the MR images based on the conductivity of the gels needs to be further investigated. Conclusions: The presented surrogates and techniques allow the customized construction of multimodality, anthropomorphic, and deformable phantoms as exemplarily shown for a pelvic phantom, which is intended to study adaptive treatment scenarios in MR-guided radiation therapy.« less

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N 6-threonylcarbamoyladenosine TcdA Function.

PubMed

Fernández, Francisco J; Gómez, Sara; Navas-Yuste, Sergio; López-Estepa, Miguel; Vega, M Cristina

2017-06-21

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N 6 -threonylcarbamoyladenosine (t 6 A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t 6 A (ct 6 A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Agarose coated spherical micro resonator for humidity measurements.

PubMed

Mallik, Arun Kumar; Liu, Dejun; Kavungal, Vishnu; Wu, Qiang; Farrell, Gerald; Semenova, Yuliya

2016-09-19

A new type of fiber optic relative humidity (RH) sensor based on an agarose coated silica microsphere resonator is proposed and experimentally demonstrated. Whispering gallery modes (WGMs) in the micro resonator are excited by evanescent coupling using a tapered fiber with ~3.3 µm waist diameter. A change in the relative humidity of the surrounding the resonator air induces changes in the refractive index (RI) and thickness of the Agarose coating layer. These changes in turn lead to a spectral shift of the WGM resonances, which can be related to the RH value after a suitable calibration. Studies of the repeatability, long-term stability, measurement accuracy and temperature dependence of the proposed sensor are carried out. The RH sensitivity of the proposed sensor depends on the concentration of the agarose gel which determines the initial thickness of the deposited coating layer. Studies of the micro- resonators with coating layers fabricated from gels with three different Agarose concentrations of 0.5%, 1.125% and 2.25 wt./vol.% showed that an increase in the initial thickness of the coating material results in an increase in sensitivity but also leads to a decrease of quality factor (Q) of the micro resonator. The highest sensitivity achieved in our experiments was 518 pm/%RH in the RH range from 30% to 70%. The proposed sensor offers the advantages of a very compact form factor, low hysteresis, good repeatability, and low cross sensitivity to temperature.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies.

PubMed

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 < 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.

A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies

PubMed Central

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 < 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. PMID:25477934

Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

PubMed Central

Xu, R; Birke, S; Carberry, S E; Geacintov, N E; Swenberg, C E; Harvey, R G

1992-01-01

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated. Images PMID:1475180

Room temperature synthesis of agarose/sol-gel glass pieces with tailored interconnected porosity.

PubMed

Cabañas, M V; Peña, J; Román, J; Vallet-Regí, M

2006-09-01

An original shaping technique has been applied to prepare porous bodies at room temperature. Agarose, a biodegradable polysaccharide, was added as binder of a sol-gel glass in powder form, yielding an easy to mold paste. Interconnected tailored porous bodies can be straightforwardly prepared by pouring the slurry into a polymeric scaffold, previously designed by stereolitography, which is subsequently eliminated by alkaline dissolution at room temperature. The so obtained pieces behave like a hydrogel with an enhanced consistency that makes them machinable and easy to manipulate. These materials generate an apatite-like layer when immersed in a simulated body fluid, indicating a potential in vivo bioactivity. The proposed method can be applied to different powdered materials to produce pieces, at room temperature, with various shapes and sizes and with tailored interconnected porosity.

Novel diffusive gradients in thin films technique to assess labile sulfate in soil.

PubMed

Hanousek, Ondrej; Mason, Sean; Santner, Jakob; Chowdhury, Md Mobaroqul Ahsan; Berger, Torsten W; Prohaska, Thomas

2016-09-01

A novel diffusive gradients in thin films (DGT) technique for sampling labile soil sulfate was developed, based on a strong basic anion exchange resin (Amberlite IRA-400) for sulfate immobilization on the binding gel. For reducing the sulfate background on the resin gels, photopolymerization was applied instead of ammonium persulfate-induced polymerization. Agarose cross-linked polyacrylamide (APA) hydrogels were used as diffusive layer. The sulfate diffusion coefficient in APA gel was determined as 9.83 × 10(-6) ± 0.35 × 10(-6) cm(2) s(-1) at 25 °C. The accumulated sulfate was eluted in 1 mol L(-1) HNO3 with a recovery of 90.9 ± 1.6 %. The developed method was tested against two standard extraction methods for soil sulfate measurement. The obtained low correlation coefficients indicate that DGT and conventional soil test methods assess differential soil sulfate pools, rendering DGT a potentially important tool for measuring labile soil sulfate.

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

PubMed

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.

Native multimer analysis of plasma and platelet von Willebrand factor compared to denaturing separation: implication for the interpretation of satellite bands.

PubMed

Hohenstein, Kurt; Griesmacher, Andrea; Weigel, Günter; Golderer, Georg; Ott, Helmut Werner

2011-06-01

Blue native electrophoresis (BNE) was applied to analyze the von Willebrand factor (vWF) multimers in their native state and to present a methodology to perform blue native electrophoresis on human plasma proteins, which has not been done before. The major difference between this method and the commonly used SDS-agarose gel electrophoresis is the lack of satellite bands in the high-resolution native gel. To further analyze this phenomenon, a second dimension was performed under denaturing conditions. Thereby, we obtained a pattern in which each protein sub-unit from the first dimension dissociates into three distinct sub-bands. These bands confirm the triplet structure, which consists of an intermediate band and two satellite bands. By introducing the second dimension, our novel method separates the triplet structure into a higher resolution than the commonly used SDS-agarose gel electrophoresis does. This helps considerably in the classification of ambiguous von Willebrand's disease subtypes. In addition, our method has the additional advantage of being able to resolve the triplet structure of platelet vWF multimers, which has not been identified previously through conventional SDS-agarose electrophoresis multimer analysis. This potential enables us to compare the triplet structure from platelet and plasmatic vWF, and may help to find out whether structural abnormalities concern the vWF molecule in the platelet itself, or whether they are due to the physiological processing of vWF shed into circulation. Owing to its resolution and sensitivity, this native separation technique offers a promising tool for the analysis and detection of von Willebrand disorder, and for the classification of von Willebrand's disease subtypes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of Proteinuria in Dogue de Bordeaux Dogs, a Breed Predisposed to a Familial Glomerulonephropathy: A Retrospective Study.

PubMed

Lavoué, Rachel; Trumel, Catherine; Smets, Pascale M Y; Braun, Jean-Pierre; Aresu, Luca; Daminet, Sylvie; Concordet, Didier; Palanché, Florence; Peeters, Dominique

2015-01-01

Dogue de Bordeaux dog has been reported to be predisposed to a familial glomerulonephropathy that displays some morphological modifications reported in focal and segmental glomerulosclerosis. Prevalence of quantitatively abnormal renal proteinuria was recently reported to be 33% in this breed. The nature of the proteinuria was assessed by sodium dodecyl sulfate-agarose gel electrophoresis and determinations of urinary markers (urinary retinol-binding protein, urinary N-acetyl-β-glucosaminidase, urinary albumin and urinary immunoglobulin G) on stored specimens. Diagnostic performances of sodium dodecyl sulfate-agarose gel electrophoresis to identify dogs with elevated urinary biomarkers were assessed. Samples from 102 adult Dogue de Bordeaux dogs (47 non-proteinuric [urine protein-to-creatinine ratio ≤ 0.2], 20 borderline-proteinuric [0.2< urine protein-to-creatinine ratio ≤ 0.5] and 35 proteinuric dogs [urine protein-to-creatinine ratio >0.5]) were used, of which 2 were suffering from familial glomerulonephropathy. The electrophoretic protein patterns, for all but one proteinuric dog, were indicative of a glomerular origin and, in all dogs, the urinary albumin concentration related to creatinine concentration and the urinary immunoglobulin G concentration related to creatinine concentration were above the upper limit of the reference interval established for the breed. Sensitivity and specificity of sodium dodecyl sulfate-agarose gel electrophoresis identifying dogs with elevated urinary albumin concentration were 94% and 92%, respectively, while diagnostic performance of sodium dodecyl sulfate-agarose gel electrophoresis in detecting dogs with elevated urinary immunoglobulin G concentration yielded sensitivity and specificity of 90% and 74%, respectively. These results suggest that all proteinuric and some borderline-proteinuric Dogue de Bordeaux dogs likely have underlying glomerular lesions and that sodium dodecyl sulfate-agarose gel electrophoresis and urinary markers might be useful to screen dogs with borderline-proteinuria. Additional investigations are warranted to assess if these findings are related to the familial glomerulonephropathy.

Ag nanoparticles agargel nanocomposites for SERS detection of cultural heritage interest pigments

NASA Astrophysics Data System (ADS)

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.

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

NASA Astrophysics Data System (ADS)

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.

Specific capture, recovery and culture of cancer cells using oriented antibody-modified polystyrene chips coated with agarose film.

PubMed

Jeong, Jiyun; Lee, Yeolin; Yoo, Yeongeun; Lee, Myung Kyu

2018-02-01

Agarose gel can be used for three dimensional (3D) cell culture because it prevents cell attachment. The dried agarose film coated on a culture plate also protected cell attachment and allowed 3D growth of cancer cells. We developed an efficient method for agarose film coating on an oxygen-plasma treated micropost polystyrene chip prepared by an injection molding process. The agarose film was modified to maleimide or Ni-NTA groups for covalent or cleavable attachment of photoactivatable Fc-specific antibody binding proteins (PFcBPs) via their N-terminal cysteine residues or 6xHis tag, respectively. The antibodies photocrosslinked onto the PFcBP-modified chips specifically captured the target cells without nonspecific binding, and the captured cells grew 3D modes on the chips. The captured cells on the cleavable antibody-modified chips were easily recovered by treatment of commercial trypsin-EDTA solution. Under fluidic conditions using an antibody-modified micropost chip, the cells were mainly captured on the micropost walls of the chip rather than on the bottom of it. The presented method will also be applicable for immobilization of oriented antibodies on various microfluidic chips with different structures. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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.

New visible and selective DNA staining method in gels with tetrazolium salts.

PubMed

Paredes, Aaron J; Naranjo-Palma, Tatiana; Alfaro-Valdés, Hilda M; Barriga, Andrés; Babul, Jorge; Wilson, Christian A M

2017-01-15

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 180 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative detection of type A staphylococcal enterotoxin by Laurell electroimmunodiffusion.

PubMed

Gasper, E; Heimsch, R C; Anderson, A W

1973-03-01

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.

Visualization of DNA molecules in time during electrophoresis

NASA Technical Reports Server (NTRS)

Lubega, Seth

1991-01-01

For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

PubMed

Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

2008-12-15

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture.

PubMed

Suzuki, Ikurou; Sugio, Yoshihiro; Moriguchi, Hiroyuki; Jimbo, Yasuhiko; Yasuda, Kenji

2004-07-01

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks.

Comparison of six simple methods for extracting ribosomal and mitochondrial DNA from Toxocara and Toxascaris nematodes.

PubMed

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.

Technical Note: Radiological properties of tissue surrogates used in a multimodality deformable pelvic phantom for MR-guided radiotherapy.

PubMed

Niebuhr, Nina I; Johnen, Wibke; Güldaglar, Timur; Runz, Armin; Echner, Gernot; Mann, Philipp; Möhler, Christian; Pfaffenberger, Asja; Jäkel, Oliver; Greilich, Steffen

2016-02-01

Phantom surrogates were developed to allow multimodal [computed tomography (CT), magnetic resonance imaging (MRI), and teletherapy] and anthropomorphic tissue simulation as well as materials and methods to construct deformable organ shapes and anthropomorphic bone models. Agarose gels of variable concentrations and loadings were investigated to simulate various soft tissue types. Oils, fats, and Vaseline were investigated as surrogates for adipose tissue and bone marrow. Anthropomorphic shapes of bone and organs were realized using 3D-printing techniques based on segmentations of patient CT-scans. All materials were characterized in dual energy CT and MRI to adapt CT numbers, electron density, effective atomic number, as well as T1- and T2-relaxation times to patient and literature values. Soft tissue simulation could be achieved with agarose gels in combination with a gadolinium-based contrast agent and NaF to simulate muscle, prostate, and tumor tissues. Vegetable oils were shown to be a good representation for adipose tissue in all modalities. Inner bone was realized using a mixture of Vaseline and K2HPO4, resulting in both a fatty bone marrow signal in MRI and inhomogeneous areas of low and high attenuation in CT. The high attenuation of outer bone was additionally adapted by applying gypsum bandages to the 3D-printed hollow bone case with values up to 1200 HU. Deformable hollow organs were manufactured using silicone. Signal loss in the MR images based on the conductivity of the gels needs to be further investigated. The presented surrogates and techniques allow the customized construction of multimodality, anthropomorphic, and deformable phantoms as exemplarily shown for a pelvic phantom, which is intended to study adaptive treatment scenarios in MR-guided radiation therapy.

Southern blotting.

PubMed

Brown, T

2001-05-01

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.

Attempt to run urinary protein electrophoresis using capillary technique.

PubMed

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.

Markers of Developmentally Regulated Programmed Cell Death and Their Analysis in Cereal Seeds.

PubMed

Domínguez, Fernando; Cejudo, Francisco Javier

2018-01-01

Programmed cell death (PCD) is a key process for the development and differentiation of multicellular organisms, which is characterized by well-defined morphological and biochemical features. These include chromatin condensation, DNA degradation and nuclear fragmentation, with nucleases and proteases playing a relevant function in these processes. In this chapter we describe methods routinely used for the analysis of hallmarks of developmentally regulated PCD in cereal seed tissues, which are based on agarose and polyacrylamide gel electrophoresis, in situ staining of DNA fragmentation, and cell-free assays of relevant enzymatic activities.

Forming giant-sized polymersomes using gel-assisted rehydration

DOE PAGES

Greene, Adrienne C.; Sasaki, Darryl Y.; Bachand, George D.

2016-05-26

Here, we present a protocol to rapidly form giant polymer vesicles ( pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

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

PubMed

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.

DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.

PubMed

Díaz-Cano, S J; Brady, S P

1997-12-01

Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.

Sex Determination Using PCR

ERIC Educational Resources Information Center

Kima, Peter E.; Rasche, Madeline E.

2004-01-01

PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

Gel Electrophoresis--The Easy Way for Students

ERIC Educational Resources Information Center

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…

Direct Quantification of Solute Diffusivity in Agarose and Articular Cartilage Using Correlation Spectroscopy.

PubMed

Shoga, Janty S; Graham, Brian T; Wang, Liyun; Price, Christopher

2017-10-01

Articular cartilage is an avascular tissue; diffusive transport is critical for its homeostasis. While numerous techniques have been used to quantify diffusivity within porous, hydrated tissues and tissue engineered constructs, these techniques have suffered from issues regarding invasiveness and spatial resolution. In the present study, we implemented and compared two separate correlation spectroscopy techniques, fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), for the direct, and minimally-invasive quantification of fluorescent solute diffusion in agarose and articular cartilage. Specifically, we quantified the diffusional properties of fluorescein and Alexa Fluor 488-conjugated dextrans (3k and 10k) in aqueous solutions, agarose gels of varying concentration (i.e. 1, 3, 5%), and in different zones of juvenile bovine articular cartilage explants (i.e. superficial, middle, and deep). In agarose, properties of solute diffusion obtained via FCS and RICS were inversely related to molecule size, gel concentration, and applied strain. In cartilage, the diffusional properties of solutes were similarly dependent upon solute size, cartilage zone, and compressive strain; findings that agree with work utilizing other quantification techniques. In conclusion, this study established the utility of FCS and RICS as simple and minimally invasive techniques for quantifying microscale solute diffusivity within agarose constructs and articular cartilage explants.

Characterization of Novel Gel Casting System to Make Complex Shaped Aluminum Oxide (Al2O3) Parts

DTIC Science & Technology

2016-03-01

investigated including systems based on starch , gelatin, protein, and agarose. Generally, all systems are too expensive for high-volume casting.13 While gel...was determined by measuring the resistance force in uniaxial compression. Therefore, the specimen was considered gelled when the resistance force was...used to lower the indenter tip at a rate of 30 mm/min by a distance of 6 mm while measuring the maximum resistance force during the indentation. At the

Sexing the Sciuridae: a simple and accurate set of molecular methods to determine sex in tree squirrels, ground squirrels and marmots.

PubMed

Gorrell, Jamieson C; Boutin, Stan; Raveh, Shirley; Neuhaus, Peter; Côté, Steeve D; Coltman, David W

2012-09-01

We determined the sequence of the male-specific minor histocompatibility complex antigen (Smcy) from the Y chromosome of seven squirrel species (Sciuridae, Rodentia). Based on conserved regions inside the Smcy intron sequence, we designed PCR primers for sex determination in these species that can be co-amplified with nuclear loci as controls. PCR co-amplification yields two products for males and one for females that are easily visualized as bands by agarose gel electrophoresis. Our method provides simple and reliable sex determination across a wide range of squirrel species. © 2012 Blackwell Publishing Ltd.

Detection of Listeria monocytogenes by using the polymerase chain reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bessesen, M.T.; Luo, Q.; Blaser, M.J.

1990-09-01

A method was developed for detection of Listeria monocytogens by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with {sup 32}P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.

21 CFR 866.4900 - Support gel.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Support gel. 866.4900 Section 866.4900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES...) Identification. A support gel for clinical use is a device that consists of an agar or agarose preparation that...

21 CFR 866.4900 - Support gel.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Support gel. 866.4900 Section 866.4900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES...) Identification. A support gel for clinical use is a device that consists of an agar or agarose preparation that...

Macrosorb Kieselguhr-agarose composite adsorbents. New tools for downstream process design and scale up. Scientific note.

PubMed

Bite, M G; Berezenko, S; Reed, F J; Derry, L

1988-08-01

Incompressible Macrosorb composite adsorbents, while retaining all the desirable properties of traditional agarose-based hydrogel media, overcome the operational limitations imposed by the use of soft hydrogels: They permit useful application of fast flow rates without restrictions on bed depth and they can be used in fluidized bed mode. Considerations which are important when contemplating scaled-up processing are discussed. A comparative cost estimate for a production process for extracting albumin from bovine serum in column equipment illustrates the various advantages which may be exploited when using a composite adsorbent in place of a conventional soft gel equivalent.

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

ERIC Educational Resources Information Center

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…

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

PubMed

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.

Problem-Solving Test: Southwestern Blotting

ERIC Educational Resources Information Center

Szeberényi, József

2014-01-01

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

PubMed

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.

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

PubMed

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.

Development of a multiplex PCR-based rapid typing method for enterohemorrhagic Escherichia coli O157 strains.

PubMed

Ooka, Tadasuke; Terajima, Jun; Kusumoto, Masahiro; Iguchi, Atsushi; Kurokawa, Ken; Ogura, Yoshitoshi; Asadulghani, Md; Nakayama, Keisuke; Murase, Kazunori; Ohnishi, Makoto; Iyoda, Sunao; Watanabe, Haruo; Hayashi, Tetsuya

2009-09-01

Enterohemorrhagic Escherichia coli O157 (EHEC O157) is a food-borne pathogen that has raised worldwide public health concern. The development of simple and rapid strain-typing methods is crucial for the rapid detection and surveillance of EHEC O157 outbreaks. In the present study, we developed a multiplex PCR-based strain-typing method for EHEC O157, which is based on the variability in genomic location of IS629 among EHEC O157 strains. This method is very simple, in that the procedures are completed within 2 h, the analysis can be performed without the need for special equipment or techniques (requiring only conventional PCR and agarose gel electrophoresis systems), the results can easily be transformed into digital data, and the genes for the major virulence markers of EHEC O157 (the stx(1), stx(2), and eae genes) can be detected simultaneously. Using this method, 201 EHEC O157 strains showing different XbaI digestion patterns in pulsed-field gel electrophoresis (PFGE) analysis were classified into 127 types, and outbreak-related strains showed identical or highly similar banding patterns. Although this method is less discriminatory than PFGE, it may be useful as a primary screening tool for EHEC O157 outbreaks.

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

PubMed

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.

Native gel analysis for RISC assembly.

PubMed

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.

Optimal processing for gel electrophoresis images: Applying Monte Carlo Tree Search in GelApp.

PubMed

Nguyen, Phi-Vu; Ghezal, Ali; Hsueh, Ya-Chih; Boudier, Thomas; Gan, Samuel Ken-En; Lee, Hwee Kuan

2016-08-01

In biomedical research, gel band size estimation in electrophoresis analysis is a routine process. To facilitate and automate this process, numerous software have been released, notably the GelApp mobile app. However, the band detection accuracy is limited due to a band detection algorithm that cannot adapt to the variations in input images. To address this, we used the Monte Carlo Tree Search with Upper Confidence Bound (MCTS-UCB) method to efficiently search for optimal image processing pipelines for the band detection task, thereby improving the segmentation algorithm. Incorporating this into GelApp, we report a significant enhancement of gel band detection accuracy by 55.9 ± 2.0% for protein polyacrylamide gels, and 35.9 ± 2.5% for DNA SYBR green agarose gels. This implementation is a proof-of-concept in demonstrating MCTS-UCB as a strategy to optimize general image segmentation. The improved version of GelApp-GelApp 2.0-is freely available on both Google Play Store (for Android platform), and Apple App Store (for iOS platform). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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.

Development and application of antibody microarray for lymphocystis disease virus detection in fish.

PubMed

Sheng, Xiuzhen; Xu, Xiaoli; Zhan, Wenbin

2013-05-01

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3-conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-L-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55μg/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 μg/ml when streptavidin-biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at -20°C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n=50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish. Copyright © 2013 Elsevier B.V. All rights reserved.

Steroid receptors analysis in human mammary tumors by isoelectric focusing in agarose.

PubMed

Bailleul, S; Gauduchon, P; Malas, J P; Lechevrel, C; Roussel, G; Goussard, J

1988-08-01

A high resolution and quantitative method for isoelectric focusing has been developed to separate the isoforms of estrogen and progesterone receptors in human mammary tumor cytosols stabilized by sodium molybdate. Agarose gels (0.5%) were used. Six samples can be analyzed on one gel in about 2 h, and 35-microliters samples are sufficient to determine the estrogen receptor isoform pattern. The constant yields and the reproducibility of data allow a quantitative analysis of these receptors. Four estrogen receptor isoforms have been observed (pI 4.7, 5.5, 6, and 6.5), isoforms with pI 4.7 and 6.5 being present in all tumors. After incubation at 28 degrees C in high ionic strength, the comparison of isoelectric focusing and high-performance size exclusion chromatography patterns of estrogen receptor confirms the oligomeric structure of the pI 4.7 isoform and suggests a monomeric structure for the pI 6.5 isoform. Under the same conditions of analysis, only one progesterone receptor isoform has been detected with pI 4.7.

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

PubMed

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.

Interaction of Electromagnetic Fields with Chondrocytes in Gel Culture

DTIC Science & Technology

1990-02-01

biosynthesis due to applied electromagnetic fields. The results suggest that both normal chondrocytes and swarm rat chondrosarcoma cells in agarose Culture...and Swarm rat chondrosarcoma cells in agarose iii culture can, under proper culture conditions, continue to synthesize ma- trix macromolecules at a...cartilagc, and in rat chondrosarcoma cells (a continuous cell line). The overt gene expression of chondrocytes results in the synthesis and deposition of a

Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

PubMed

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.

Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.

PubMed

Zeng, Z; Clark, S M; Mathies, R A; Glazer, A N

1997-10-01

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

Structural features of the nonionic surfactants stabilizing long-lived bubble nuclei

NASA Technical Reports Server (NTRS)

Darrigo, J. S.

1980-01-01

A study of the effects of various electrolytes and one organic compound on bubble production in agarose gels is presented. Several preparations of ultrapure agarose were compared for 42 electrolytes and phenol to identify trends in bubble formation. The anion and cation sequences of bubble suppression are similar to processes for salting out of nonionic surfactants. The reduction of bubble number by polyvalent ions and 1% phenol suggests that the polar portions of these nonionic surfactants represent amide groups. The evidence for amide groups is consistent with the relative positions of Mg(2+) in all cation sequences; this result makes it unlikely that either linkages contribute to the hydrophilicity of the nonionic surfactants stabilizing bubble nuclei in the different aqueous gels tested.

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

PubMed

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

1990-06-01

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 invasive plasmid. The application of PCR as a rapid method to detect enteroinvasive bacteria in foods was tested by inoculating lettuce with 10(4) S. flexneri cells per g in shigella broth base. Plasmid DNA was isolated from cultures of inoculated and uninoculated lettuce in broth after 0, 4, and 24 h of incubation. With the PCR, the 760-bp fragment was generated only from lettuce inoculated with S. flexneri, as shown by gel electrophoresis and confirmed both by Southern blotting and by nucleotide sequencing of the amplified region. Because the isolation of plasmid DNA, the performance of PCR, and gel electrophoresis all can be completed in 6 to 7 h, invasive enteric bacteria can be detected in less than 1 day.

Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis.

PubMed

Robinson, Nicholas P

2013-01-01

Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native-native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

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

PubMed

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.

Potential application of immobilized streptokinase extracted from Streptococcus equinus VIT_VB2.

PubMed

Vaishnavi, B; Subathra Devi, C

2017-11-26

Streptokinase purified from Streptococcus equinus VIT_VB2 isolated from bovine milk sample was immobilized in various solid supports namely entrapment in agarose gel, calcium alginate beads and gelatin gel by cross-linking with formaldehyde. Immobilization of streptokinase in calcium alginate beads showed maximum efficiency (81.8 ± 1.06%) when compared with entrapment with agarose gel (55.6 ± 2.17%) and cross-linked gelatin formaldehyde gel (71.0 ± 1.54%). The purified SK activity was expressed maximum in calcium alginate (1%) and gelatin gel (0.25%) with 1292.68 ± 1.33 and 1121.9 ± 1.2 U mL -1 , respectively. Similarly, SK entrapped in gelatin gel and calcium alginate showed maximum in vitro blood clot lysis activity with 77.67 ± 2.64% and 76.16 ± 2.72%, respectively. The immobilized SK in gelatin gel showed complete clot lysis within 15 min; hence, this application of the study could be used in the treatment of superficial thrombophlebitis, phlebitis, and venous thrombosis. These beads were used for three repeated cycles to check the conversion of substrates into their products, and we concluded that SK can be immobilized in the suitable matrices. Therefore, this helps in the drug-delivery strategies in highly efficient way, moreover, economically competent process in the pharmaceutics.

A Novel Adjuvant-Solution Layer Strategy for Improving the Efficacy of Cryosurgery.

PubMed

Ramajayam, K K; Kumar, A; Sarangi, S K; Thirugnanam, A

During cryosurgery, studies reported earlier suggest increased destruction inside the tumour due to adjuvants or the prevention of damage to neighbouring healthy tissue through different methods. This study advocates a novel strategy that increases the freezing inside agarose gel phantoms by addition of glycine and limits the freezing to the desired location using a perfluorohexane layer during cryosurgery. Cryosurgery of glycine containing gels is carried out with and without perfluorohexane layer and the thermal history is measured using K-type thermocouples connected to a data acquisition system. The presence of glycine causes increased freezing during cryosurgery with an ice ball depth of 16 mm, while with a perfluorohexane layer at this gel interface, this depth is 13 mm, indicating the ability of this layer to limit freezing. It can be concluded that glycine addition results in substantial temperature decrease and perfluorohexane layer insulates the glycine gel effectively during cryosurgical cooling.

Competitor internal standards for quantitative detection of mycoplasma DNA.

PubMed

Sidhu, M K; Rashidbaigi, A; Testa, D; Liao, M J

1995-05-01

Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumoniae. To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.

groEL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop-mediated isothermal amplification assay.

PubMed

Siddique, M P; Jang, W J; Lee, J M; Ahn, S H; Suraiya, S; Kim, C H; Kong, I S

2017-08-01

A groEL gene-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Vibrio parahaemolyticus in contaminated seafood and water. The assay was optimized and conducted at 63°C for 40 min using Bacillus stearothermophilus (Bst) DNA polymerase, large fragment. Amplification was analysed via multiple detection methods, including opacity, formation of white precipitate, DNA intercalating dyes (ethidium bromide and SYBR Green I), metal ion-binding indicator dye, calcein, and 2% agarose gel electrophoresis. A characteristic ladder-like band pattern on agarose gel and the desired colour changes when using different dyes were observed in positive cases, and these were species-specific for V. parahaemolyticus when compared with other closely related Vibrio spp. The limit of detection (LoD) of this assay was 100 fg per reaction, 100-fold higher than that for conventional polymerase chain reaction (PCR). When tested on artificially contaminated seafood and seawater, the LoDs of the LAMP assay were 120 and 150 fg per reaction respectively, and those of conventional PCR were 120 and 150 pg per reaction respectively. Based on our results, the groEL gene-based LAMP assay is rapid, specific, sensitive, and reliable for detecting V. parahaemolyticus, and it could be used in field diagnosis. The loop-mediated isothermal amplification (LAMP) assay using groEL gene (an abundant, highly conserved gene and member of the groESL chaperone gene family) provided rapid, species-specific and highly sensitive method for detecting Vibrio parahaemolyticus, the leading causal agent of seafood-borne diseases worldwide. Moreover, groEL LAMP revealed high efficiency than conventional PCR assay for V. parahaemolyticus using template both from pure culture and artificially contaminated seafood and water, which indicated the applicability in the field and environmental screening purpose for the organism. © 2017 The Society for Applied Microbiology.

Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

ERIC Educational Resources Information Center

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.…

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis

PubMed Central

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

Investigations on gel forming media use in low gravity bioseparations research

NASA Technical Reports Server (NTRS)

Todd, Paul; Szlag, David C.; Plank, Lindsay D.; Delcourt, Scott G.; Kunze, M. Elaine

1989-01-01

Research on gelling media and conditions suitable for the preservation of the spatial configuration of cell suspensions and macromolecular solutions after separation in free fluid during low gravity experiments is presented. The examples studied included free electrophoresis of cells in a cylindrical column and two-phase aqueous polymer separation. Microgravity electrophoresis experiments were simulated by separating model cell types (animal or human) in a vertical density gradient containing low-conductivity buffer, 1.7-6.5 percent Ficoll, 6.8-5.0 percent sucrose, and 1 percent SeaPrep low-melting temperature agarose. Upon cooling, a gel formed in the column and cells could be captured at the forming locations. Two-phase extraction experiments were simulated using two-polymer solutions in which phase separation occurs in normal saline at temperatures compatible with cell viability and in which one or both phases form a gel upon cooling. Suitable polymers included commercial agaroses (1-2 percent), maltodextrin (5-7 percent), and gelatin (5-20 percent).

Agarose gels

NASA Astrophysics Data System (ADS)

2016-11-01

Discovered in 17th-century Japan, agar is a jelly-like substance obtained by boiling algae, and it is widely used as a gelling agent for desserts in Japanese, Indian, Philippine and Vietnamese cuisine.

Comparison of Two Capillary Gel Electrophoresis Systems for Clostridium difficile Ribotyping, Using a Panel of Ribotype 027 Isolates and Whole-Genome Sequences as a Reference Standard

PubMed Central

Xiao, Meng; Kong, Fanrong; Jin, Ping; Wang, Qinning; Xiao, Kelin; Jeoffreys, Neisha; James, Gregory

2012-01-01

PCR ribotyping is the most commonly used Clostridium difficile genotyping method, but its utility is limited by lack of standardization. In this study, we analyzed four published whole genomes and tested an international collection of 21 well-characterized C. difficile ribotype 027 isolates as the basis for comparison of two capillary gel electrophoresis (CGE)-based ribotyping methods. There were unexpected differences between the 16S-23S rRNA intergenic spacer region (ISR) allelic profiles of the four ribotype 027 genomes, but six bands were identified in all four and a seventh in three genomes. All seven bands and another, not identified in any of the whole genomes, were found in all 21 isolates. We compared sequencer-based CGE (SCGE) with three different primer pairs to the Qiagen QIAxcel CGE (QCGE) platform. Deviations from individual reference/consensus band sizes were smaller for SCGE (0 to 0.2 bp) than for QCGE (4.2 to 9.5 bp). Compared with QCGE, SCGE more readily distinguished bands of similar length (more discriminatory), detected bands of larger size and lower intensity (more sensitive), and assigned band sizes more accurately and reproducibly, making it more suitable for standardization. Specifically, QCGE failed to identify the largest ISR amplicon. Based on several criteria, we recommend the primer set 16S-USA/23S-USA for use in a proposed standard SCGE method. Similar differences between SCGE and QCGE were found on testing of 14 isolates of four other C. difficile ribotypes. Based on our results, ISR profiles based on accurate sequencer-based band lengths would be preferable to agarose gel-based banding patterns for the assignment of ribotypes. PMID:22692737

Kilo-sequencing: an ordered strategy for rapid DNA sequence data acquisition.

PubMed Central

Barnes, W M; Bevan, M

1983-01-01

A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitro procedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA, may be purified by electrophoresis alive on agarose gels. Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA. Images PMID:6298723

Generation of strip-format fibrin-based engineered heart tissue (EHT).

PubMed

Schaaf, Sebastian; Eder, Alexandra; Vollert, Ingra; Stöhr, Andrea; Hansen, Arne; Eschenhagen, Thomas

2014-01-01

This protocol describes a method for casting fibrin-based engineered heart tissue (EHT) in standard 24-well culture dishes. In principle, a hydrogel tissue engineering method requires cardiomyocytes, a liquid matrix that forms a gel, a casting mold, and a device that keeps the developing tissue in place. This protocol refers to neonatal rat heart cells as the cell source; the matrix of choice is fibrin, and the tissues are generated in rectangular agarose-casting molds (12 × 3 × 3 mm) prepared in standard 24-well cell culture dishes, in which a pair of flexible silicone posts is suspended from above. A master mix of freshly isolated cells, medium, fibrinogen, and thrombin is pipetted into the casting mold and, over a period of 2 h, polymerizes and forms a fibrin cell block around two silicone posts. Silicone racks holding four pairs of silicone posts each are used to transfer the fresh fibrin cell blocks into new 24-well dishes with culture medium. Without further handling, the cells start to remodel the fibrin gel, form contacts with each other, elongate, and condense the gel to approximately ¼ of the initial volume. Spontaneous and rhythmic contractions start after 1 week. EHTs are viable and relatively stable for several weeks in this format and can be subjected to repeated measurements of contractile function and final morphological and molecular analyses.

DNA-based differentiation of the Ecuadorian cocoa types CCN-51 and Arriba based on sequence differences in the chloroplast genome.

PubMed

Herrmann, Luise; Haase, Ilka; Blauhut, Maike; Barz, Nadine; Fischer, Markus

2014-12-17

Two cocoa types, Arriba and CCN-51, are being cultivated in Ecuador. With regard to the unique aroma, Arriba is considered a fine cocoa type, while CCN-51 is a bulk cocoa because of its weaker aroma. Because it is being assumed that Arriba is mixed with CCN-51, there is an interest in the analytical differentiation of the two types. Two methods to identify CCN-51 adulterations in Arriba cocoa were developed on the basis of differences in the chloroplast DNA. On the one hand, a different repeat of the sequence TAAAG in the inverted repeat region results in a different length of amplicons for the two cocoa types, which can be detected by agarose gel electrophoresis, capillary gel electrophoresis, and denaturing high-performance liquid chromatography. On the other hand, single nucleotide polymorphisms (SNPs) between the CCN-51 and Arriba sequences represent restriction sites, which can be used for restriction fragment length polymorphism analysis. A semi-quantitative analysis based on these SNPs is feasible. A method for an exact quantitation based on these results is not realizable. These sequence variations were confirmed for a comprehensive cultivar collection of Arriba and CCN-51, for both bean and leaf samples.

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

NASA Astrophysics Data System (ADS)

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.

High performance gel imaging with a commercial single lens reflex camera

NASA Astrophysics Data System (ADS)

Slobodan, J.; Corbett, R.; Wye, N.; Schein, J. E.; Marra, M. A.; Coope, R. J. N.

2011-03-01

A high performance gel imaging system was constructed using a digital single lens reflex camera with epi-illumination to image 19 × 23 cm agarose gels with up to 10,000 DNA bands each. It was found to give equivalent performance to a laser scanner in this high throughput DNA fingerprinting application using the fluorophore SYBR Green®. The specificity and sensitivity of the imager and scanner were within 1% using the same band identification software. Low and high cost color filters were also compared and it was found that with care, good results could be obtained with inexpensive dyed acrylic filters in combination with more costly dielectric interference filters, but that very poor combinations were also possible. Methods for determining resolution, dynamic range, and optical efficiency for imagers are also proposed to facilitate comparison between systems.

Acetylcholinesterase of the Sand Fly, Phlebotomus papatasi (Scopoli): Construction, Expression and Biochemical Properties of the G119S Orthologous Mutant

DTIC Science & Technology

2014-12-10

synthesis and agarose gel electrophoresis were as previously described [18]. Anticholinesterases as probes of enzyme function The experimental... anticholinesterases used in this study for enzyme characterization are shown in Figure 1. They were synthesized and purified via established methods [29-31] and had...structures and names of experimental anticholinesterases used in this study. Bold numbers beside the names denote the compounds as presented in the text. For

The effects of metal ions on the DNA damage induced by hydrogen peroxide.

PubMed

Kobayashi, S; Ueda, K; Komano, T

1990-01-01

The effects of metal ions on DNA damage induced by hydrogen peroxide were investigated using two methods, agarose-gel electrophoretic analysis of supercoiled DNA and sequencing-gel analysis of single end-labeled DNA fragments of defined sequences. Hydrogen peroxide induced DNA damage when iron or copper ion was present. At least two classes of DNA damage were induced, one being direct DNA-strand cleavage, and the other being base modification labile to hot piperidine. The investigation of the damaged sites and the inhibitory effects of radical scavengers revealed that hydroxyl radical was the species which attacked DNA in the reaction of H2O2/Fe(II). On the other hand, two types of DNA damage were induced by H2O2/Cu(II). Type I damage was predominant and inhibited by potassium iodide, but type II was not. The sites of the base-modification induced by type I damage were similar to those by lipid peroxidation products and by ascorbate in the presence of Cu(II), suggesting the involvement of radical species other than free hydroxyl radical in the damaging reactions.

Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G) by Different Strategies

PubMed Central

Mendes, Adriano A.; Freitas, Larissa; de Carvalho, Ana Karine F.; de Oliveira, Pedro C.; de Castro, Heizir F.

2011-01-01

The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g−1 of support) was achieved when the lipase was immobilized on epoxy-SiO2-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g−1 of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g−1 of gel, and the highest activity (68.8 ± 2.70 IU·g−1 of support) was obtained when 20 mg of protein·g−1 was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO2-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase. PMID:21811674

Lymphocyte receptors for pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clark, C.G.; Armstrong, G.D.

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, andmore » 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.« less

Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.

PubMed

Nair, Smita; Manimekalai, Ramaswamy; Ganga Raj, Palliyath; Hegde, Vinayaka

2016-07-01

The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.

High-quality cell block preparation from scraping of conventional cytology slide: a technical report on a modified cytoscrape cell block technique.

PubMed

Choi, Y I; Jakhongir, M; Choi, S J; Kim, L; Park, I S; Han, J Y; Kim, J M; Chu, Y C

2016-12-01

Immunocytochemistry (ICC) on formalin-fixed paraffin embedded cell blocks is an ancillary tool commonly recruited for differential diagnoses of fine needle aspiration cytology (FNAC) samples. However, the quality of conventional cell blocks in terms of adequate cellularity and evenness of distribution of cytologic material is not always satisfactory for ICC. We introduce a modified agarose-based cytoscrape cell block (CCB) technique that can be effectively used for the preparation of cell blocks from scrapings of conventional FNAC slides. A decoverslipped FNAC slide was mounted with a small amount of water. The cytological material was scraped off the slide into a tissue mold by scraping with a cell scraper. The cytoscrape material was pelleted by centrifugation and pre-embedded in ultra-low gelling temperature agarose and then re-embedded in conventional agarose. The final agarose gel disk was processed and embedded in paraffin. The quality of the ICC on the CCB sections was identical to that of the immunohistochemical stains on histological sections. By scrapping and harvesting the entirety of the cytological material off the cytology slide into a compact agarose cell button, we could avoid the risk of losing diagnostic material during the CCB preparation. This modified CCB technique enables concentration and focusing of minute material while maintaining the entire amount of the cytoscrape material on the viewing spot of the CCB sections. We believe this technique can be effectively used to improve the level of confidence in diagnosis of FNAC especially when the FNAC slides are the only sample available.

Development of bufferless gel electrophoresis chip for easy preparation and rapid DNA separation.

PubMed

Oleksandrov, Sergiy; Aman, Abdurazak; Lim, Wanyoung; Kim, Younghee; Bae, Nam Ho; Lee, Kyoung G; Lee, Seok Jae; Park, Sungsu

2018-02-01

This work presents a handy, fast, and compact bufferless gel electrophoresis chip (BGEC), which consists of precast agarose gel confined in a disposable plastic body with electrodes. It does not require large volumes of buffer to fill reservoirs, or the process of immersing the gel in the buffer. It withstands voltages up to 28.4 V/cm, thereby allowing DNA separation within 10 min with a similar separation capability to the standard gel electrophoresis. The results suggest that our BGEC is highly suitable for in situ gel electrophoresis in forensic, epidemiological settings and crime scenes where standard gel electrophoresis equipment cannot be brought in while quick results are needed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of Agar Gel Strength and Fat on Oral Breakdown, Volatile Release, and Sensory Perception Using in Vivo and in Vitro Systems.

PubMed

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.

Chemical improvement of chitosan-modified beads for the immobilization of Enterococcus faecium DBFIQ E36 L-arabinose isomerase through multipoint covalent attachment approach.

PubMed

Manzo, Ricardo M; de Sousa, Marylane; Fenoglio, Cecilia L; Gonçalves, Luciana Rocha Barro; Mammarella, Enrique J

2015-10-01

D-tagatose is produced from D-galactose by the enzyme L-arabinose isomerase (L-AI) in a commercially viable bioprocess. An active and stable biocatalyst was obtained by modifying chitosan gel structure through reaction with TNBS, D-fructose or DMF, among others. This led to a significant improvement in L-AI immobilization via multipoint covalent attachment approach. Synthetized derivatives were compared with commercial supports such as Eupergit(®) C250L and glyoxal-agarose. The best chitosan derivative for L-AI immobilization was achieved by reacting 4 % (w/v) D-fructose with 3 % (w/v) chitosan at 50 °C for 4 h. When compared to the free enzyme, the glutaraldehyde-activated chitosan biocatalyst showed an apparent activity of 88.4 U g (gel) (-1) with a 211-fold stabilization factor while the glyoxal-agarose biocatalyst gave an apparent activity of 161.8 U g (gel) (-1) with an 85-fold stabilization factor. Hence, chitosan derivatives were comparable to commercial resins, thus becoming a viable low-cost strategy to obtain high active L-AI insolubilized derivatives.

Development of a bi-functional silica monolith for electro-osmotic pumping and DNA clean-up/extraction using gel-supported reagents in a microfluidic device.

PubMed

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.

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization.

PubMed

Jenkins, Frank J; Kerr, Charles M; Fouquerel, Elise; Bovbjerg, Dana H; Opresko, Patricia L

2017-07-10

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern 1 , 2 . Here, we present a detailed description of this procedure, with some modifications.

One-step separation and purification of hydrolysable tannins from Geranium wilfordii Maxim by adsorption chromatography on cross-linked 12% agarose gel.

PubMed

Liu, Dan; Ma, Yan; Wang, Ye; Su, Zhiguo; Gu, Ming; Janson, Jan-Christer

2011-05-01

The hydrolysable tannins corilagin and geraniin, the major active components of the traditional Chinese medicine Geranium wilfordii Maxim, have been separated and purified from crude extracts in one step by adsorption chromatography on cross-linked 12% agarose gel (Superose 12 10/300 GL). The separation was achieved by gradient elution using mobile phase A composed of 5% ethanol and 5% acetic acid and mobile phase B composed of 30% ethanol and 30% acetic acid. The gradients were composed as follows: 0-240 mL, 0-25% B; 240-480 mL, 25-40% B; after 480 mL, 100% B. The purities of the collected corilagin and geraniin were 92.4 and 87.2%, and the corresponding yields were 88.0 and 76.8%, respectively. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microwell hybridization assay for detection of PCR products from Mycobacterium tuberculosis complex and the recombinant Mycobacterium smegmatis strain 1008 used as an internal control.

PubMed Central

Kox, L F; Noordhoek, G T; Kunakorn, M; Mulder, S; Sterrenburg, M; Kolk, A H

1996-01-01

A microwell hybridization assay was developed for the detection of the PCR products from both Mycobacterium tuberculosis complex bacteria and the recombinant Mycobacterium smegmatis strain 1008 that is used as an internal control to monitor inhibition in the PCR based on the M. tuberculosis complex-specific insertion sequence IS6110. The test is based on specific detection with digoxigenin-labeled oligonucleotide probes of biotinylated PCR products which are captured in a microtiter plate coated with streptavidin. The captured PCR products are hybridized separately with two probes, one specific for the PCR product from IS6110 from M. tuberculosis complex and the other specific for the PCR fragment from the modified IS6110 fragment from the recombinant M. smegmatis 1008. The microwell hybridization assay discriminates perfectly between the two types of amplicon. The amount of PCR product that can be detected by this assay is 10 times less than that which can be detected by agarose gel electrophoresis. The test can be performed in 2 h. It is much faster and less laborious than Southern blot hybridization. Furthermore, the interpretation of results is objective. The assay was used with 172 clinical samples in a routine microbiology laboratory, and the results were in complete agreement with those of agarose gel electrophoresis and Southern blot hybridization. PMID:8862568

[Single-molecule detection and characterization of DNA replication based on DNA origami].

PubMed

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Mechanics of a fiber network within a non-fibrillar matrix: model and comparison with collagen-agarose co-gels

PubMed Central

Lake, Spencer P.; Hadi, Mohammad F.; Lai, Victor K.; Barocas, Victor H.

2013-01-01

While collagen is recognized as the predominant mechanical component of soft connective tissues, the role of the non-fibrillar matrix (NFM) is less well understood. Even model systems, such as the collagen-agarose co-gel, can exhibit complex behavior, making it difficult to identify relative contributions of specific tissue constituents. In the present study, we developed a two-component microscale model of collagen-agarose tissue analogs and used it to elucidate the interaction between collagen and NFM in uniaxial tension. Collagen fibers were represented with Voronoi networks, and the NFM was modeled as a neo-Hookean solid. Model predictions of total normal stress and Poisson’s ratio matched experimental observations well (including high Poisson’s values of ~3), and the addition of NFM led to composition-dependent decreases in volume change and increases in fiber stretch. Because the NFM was more resistant to volume change than the fiber network, extension of the composite led to pressurization of the NFM. Within a specific range of parameter values (low shear modulus and moderate Poisson’s ratio), the magnitude of the reaction force decreased relative to this pressurization component resulting in a negative (compressive) NFM stress in the loading direction, even though the composite tissue was in tension. PMID:22565816

Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

NASA Astrophysics Data System (ADS)

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.

Threading dynamics of a polymer through parallel pores: Potential applications to DNA size separation

NASA Astrophysics Data System (ADS)

Åkerman, Björn

1997-04-01

DNA orientation measurements by linear dichroism (LD) spectroscopy and single molecule imaging by fluorescence microscopy are used to investigate the effect of DNA size (71-740 kilo base pairs) and field strength E (1-5.9 V/cm) on the conformation dynamics during the field-driven threading of DNA molecules through a set of parallel pores in agarose gels, with average pore radii between 380 Å and 1400 Å. Locally relaxed but globally oriented DNA molecules are subjected to a perpendicular field, and the observed LD time profile is compared with a recent theory for the threading [D. Long and J.-L. Viovy, Phys. Rev. E 53, 803 (1996)] which assumes the same initial state. As predicted the DNA is driven by the ends into a U-form, leading to an overshoot in the LD. The overshoot-time scales as E-(1.2-1.4) as predicted, but grows more slowly with DNA size than the predicted linear dependence. For long molecules loops form initially in the threading process but are finally consumed by the ends, and the process of transfer of DNA segments, from the loops to the arms of the U, leads to a shoulder in the LD as predicted. The critical size below which loops do not form (as indicated by the LD shoulder being absent) is between 71 and 105 kbp (0.5% agarose, 5.9 V/cm), and considerably larger than predicted because in the initial state the DNA molecules are housed in gel cavities with effective pore sizes about four times larger than the average pore size. From the data, the separation of DNA by exploiting the threading dynamics in pulsed fields [D. Long et al., CR Acad. Sci. Paris, Ser. IIb 321, 239 (1995)] is shown to be feasible in principle in an agarose-based system.

Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yinfa, Ma.

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column'' detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal of this dissertation to present a new detection method for TLC -- indirect fluorometric detection method. This detection technique is universal sensitive, nondestructive, and simple. This will bemore » described in detail from Sections 1 through Section 5. Section 1 and 3 describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. In Section 2, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. In Section 4, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages. A laser-based indirect fluorometric detection technique in TLC is developed in Section 5. Section 6 is totally different from Sections 1 through 5. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. 262 refs.« less

Problem-solving test: Southwestern blotting.

PubMed

Szeberényi, József

2014-01-01

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

A Small Stem Loop Structure Of The Ebola Virus Trailer Is Essential For Replication And Interacts With Heat Shock Protein A8

DTIC Science & Technology

2016-12-02

agarose gel electrophoresis TR-16-205 Nucleic Acids Research, 2016 3 (Seakem GTG , Sigma-Aldrich) and purified using the QI- Aquick Gel Extraction Kit... gtg +cga 1923-1938 3′ LNA2 +caa+aaa+ga+aa+gaa+gaa 3E-5E eGFP, 3E-5E plasmid containing enhanced green fluorescent protein; aiSHAPE, antisense-interfered

Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

PubMed

Pelaez, Daniel; Huang, Chun-Yuh Charles; Cheung, Herman S

2009-01-01

Mechanical loading has long been shown to modulate cartilage-specific extracellular matrix synthesis. With joint motion, cartilage can experience mechanical loading in the form of compressive, tensile or shearing load, and hydrostatic pressure. Recent studies have demonstrated the capacity of unconfined cyclic compression to induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose culture. However, the use of a nonbiodegradable material such as agarose limits the applicability of these constructs. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Our results show that cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days. This study demonstrates the suitability of fibrin gel for supporting the cyclic compression-induced chondrogenesis of mesenchymal stem cells.

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

NASA Astrophysics Data System (ADS)

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.

Metabolic studies of mammalian cells by 31P-NMR using a continuous perfusion technique.

PubMed

Knop, R H; Chen, C W; Mitchell, J B; Russo, A; McPherson, S; Cohen, J S

1984-07-20

Levels of ATP and Pi in metabolically active Chinese hamster lung fibroblasts were monitored noninvasively by 31P-NMR over many hours and under a variety of conditions. The cells were embedded in a matrix of agarose gel in the form of fine threads which were continuously perfused in a standard NMR tube. The small diameter of the thread allows rapid diffusion of metabolites and drugs into the cells. The changes in ATP and Pi levels were followed as a function of time in response to perfusion with a glucose-containing medium, with isotonic saline and with a medium containing 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. This gel-thread perfusion method should enable routine NMR studies of cellular metabolism, and may have other potential biological applications.

Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments.

PubMed Central

Gray, A J; Beecher, D E; Olson, M V

1984-01-01

A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments. Images PMID:6320097

Refractive-index-matched hydrogel materials for measuring flow-structure interactions

NASA Astrophysics Data System (ADS)

Byron, Margaret L.; Variano, Evan A.

2013-02-01

In imaging-based studies of flow around solid objects, it is useful to have materials that are refractive-index-matched to the surrounding fluid. However, materials currently in use are usually rigid and matched to liquids that are either expensive or highly viscous. This does not allow for measurements at high Reynolds number, nor accurate modeling of flexible structures. This work explores the use of two hydrogels (agarose and polyacrylamide) as refractive-index-matched models in water. These hydrogels are inexpensive, can be cast into desired shapes, and have flexibility that can be tuned to match biological materials. The use of water as the fluid phase allows this method to be implemented immediately in many experimental facilities and permits investigation of high-Reynolds-number phenomena. We explain fabrication methods and present a summary of the physical and optical properties of both gels, and then show measurements demonstrating the use of hydrogel models in quantitative imaging.

Convective polymer assembly for the deposition of nanostructures and polymer thin films on immobilized particles.

PubMed

Richardson, Joseph J; Björnmalm, Mattias; Gunawan, Sylvia T; Guo, Junling; Liang, Kang; Tardy, Blaise; Sekiguchi, Shota; Noi, Ka Fung; Cui, Jiwei; Ejima, Hirotaka; Caruso, Frank

2014-11-21

We report the preparation of polymer particles via convective polymer assembly (CPA). Convection is used to move polymer solutions and cargo through an agarose gel that contains immobilized template particles. This method both coats and washes the particles in a process that is amenable to automation, and does not depend on passive diffusion or electrical currents, thus facilitating incorporation of fragile and nanoscale objects, such as liposomes and gold nanoparticles, into the thin polymer films. Template dissolution leads to the formation of stable polymer particles and capsules.

Double-tagged fluorescent bacterial bioreporter for the study of polycyclic aromatic hydrocarbon diffusion and bioavailability.

PubMed

Tecon, Robin; Binggeli, Olivier; van der Meer, Jan R

2009-09-01

Bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), ubiquitous contaminants from oil and coal, is typically limited by poor accessibility of the contaminant to the bacteria. In order to measure PAH availability in complex systems, we designed a number of diffusion-based assays with a double-tagged bacterial reporter strain Burkholderia sartisoli RP037-mChe. The reporter strain is capable of mineralizing phenanthrene (PHE) and induces the expression of enhanced green fluorescent protein (eGFP) as a function of the PAH flux to the cell. At the same time, it produces a second autofluorescent protein (mCherry) in constitutive manner. Quantitative epifluorescence imaging was deployed in order to record reporter signals as a function of PAH availability. The reporter strain expressed eGFP proportionally to dosages of naphthalene or PHE in batch liquid cultures. To detect PAH diffusion from solid materials the reporter cells were embedded in 2 cm-sized agarose gel patches, and fluorescence was recorded over time for both markers as a function of distance to the PAH source. eGFP fluorescence gradients measured on known amounts of naphthalene or PHE served as calibration for quantifying PAH availability from contaminated soils. To detect reporter gene expression at even smaller diffusion distances, we mixed and immobilized cells with contaminated soils in an agarose gel. eGFP fluorescence measurements confirmed gel patch diffusion results that exposure to 2-3 mg lampblack soil gave four times higher expression than to material contaminated with 10 or 1 (mg PHE) g(-1).

DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.

PubMed

Colasuonno, Pasqualina; Incerti, Ornella; Lozito, Maria Luisa; Simeone, Rosanna; Gadaleta, Agata; Blanco, Antonio

2016-02-17

Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations. In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85% ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, β-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes. We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions. The availability of this tool together with TILLING technique will expand the polymorphisms in candidate genes of agronomically important traits in wheat.

A rapid high-resolution method for resolving DNA topoisomers.

PubMed

Mitchenall, Lesley A; Hipkin, Rachel E; Piperakis, Michael M; Burton, Nicolas P; Maxwell, Anthony

2018-01-16

Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets.

Real-time nested multiplex PCR for the detection of herpes simplex virus types 1 and 2 and varicella zoster virus.

PubMed

O'Neill, Hugh J; Wyatt, Dorothy E; Coyle, Peter V; McCaughey, Conall; Mitchell, Frederick

2003-12-01

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols. Copyright 2003 Wiley-Liss, Inc.

Single bead-based electrochemical biosensor.

PubMed

Liu, Changchun; Schrlau, Michael G; Bau, Haim H

2009-12-15

A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensor's working electrode consists of an electrochemically etched platinum wire, with a nominal diameter of 25 microm, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). The sealing process does not require any epoxy or glue. A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized bead eliminates the tedious and complicated surface functionalization process that is often the bottleneck in the development of electrochemical biosensors. We report on the use of a biotin agarose bead-based, micropipette, electrochemical (Bio-BMP) biosensor to monitor H(2)O(2) concentration and the use of a streptavidin bead-based, micropipette, electrochemical (SA-BMP) biosensor to detect DNA amplicons. The Bio-BMP biosensor's response increased linearly as the H(2)O(2) concentration increased in the range from 1 x 10(-6) to 1.2 x10(-4)M with a detection limit of 5 x 10(-7)M. The SA-BMP was able to detect the amplicons of 1pg DNA template of B. Cereus bacteria, thus providing better detection sensitivity than conventional gel-based electropherograms.

Molecular – genetic variance of RH blood group system within human population of Bosnia and Herzegovina

PubMed Central

Lasić, Lejla; Lojo-Kadrić, Naida; Silajdžić, Elma; Pojskić, Lejla; Hadžiselimović, Rifat; Pojskić, Naris

2013-01-01

There are two major theories for inheritance of Rh blood group system: Fisher – Race theory and Wiener theory. Aim of this study was identifying frequency of RHDCE alleles in Bosnian – Herzegovinian population and introduction of this method in screening for Rh phenotype in B&H since this type of analysis was not used for blood typing in B&H before. Rh blood group was typed by Polymerase Chain Reaction, using the protocols and primers previously established by other authors, then carrying out electrophoresis in 2-3% agarose gel. Percentage of Rh positive individuals in our sample is 84.48%, while the percentage of Rh negative individuals is 15.52%. Inter-rater agreement statistic showed perfect agreement (K=1) between the results of Rh blood system detection based on serological and molecular-genetics methods. In conclusion, molecular – genetic methods are suitable for prenatal genotyping and specific cases while standard serological method is suitable for high-throughput of samples. PMID:23448604

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology.

PubMed

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.

DNA Camouflage

DTIC Science & Technology

2016-01-08

Kan), and pBZ51 + pBZ52 (selected on Amp ) were grown overnight, and plasmid DNA was extracted and run on a 1% agarose gel. Cells co-transformed with...pBZ51 and pBZ52 were able to stably maintain both plasmids under Amp selection. SI Fig. 10 SI

Practical Molecular Biology for Students: An Integrated Approach to Teaching Basic Techniques.

ERIC Educational Resources Information Center

Hames, B. David; And Others

1990-01-01

An activity that introduces students to the correct handling of bacterial recombinants, antibiotic sensitivity testing, insertional inactivation, plasmid DNA isolation, restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, hybridization, and autoradiography is presented. A list of needed materials, procedures, safety…

Interaction of electromagnetic fields with chondrocytes in gel culture

NASA Astrophysics Data System (ADS)

Grodzinsky, Alan J.; Buschmann, Michael D.; Gluzband, Yehezkiel A.

1992-01-01

The specific objectives of this research period were: (1) to quantify the effect of applied electric fields on chondrocyte metabolism, using a range of stimulation frequencies and amplitudes; (2) to compare the chondrocyte biosynthetic response to applied fields at early times in agarose gel culture before an extracellular matrix has accumulated and at later times after significant deposition of matrix around and between the cells; and (3) to begin to interpret the biosynthetic response to applied fields in terms of models of physical mechanisms. The results of these studies suggest that electric fields applied to chondrocytes in agarose can modulate the synthesis of proteoglycans and protein constituents. Biosynthesis may be inhibited or stimulated depending on the amplitude of the applied current density. In addition, the presence of extracellular matrix may enhance the ability of normal chondrocytes and cells in intact cartilage to respond to electric fields, although the presence of matrix was not required for the stimulatory response to be observed with Swarm rat chondrosarcoma cells.

Analysis of splicing complexes on native gels.

PubMed

Ares, Manuel

2013-10-01

Splicing requires a complex set of ATP-dependent macromolecular associations that lead to the rearrangement of just a few covalent bonds in the pre-mRNA substrate. Seeing only the covalent bonds breaking and forming is seeing only a very small part of the process. Analysis of native splicing complexes into which the radiolabeled substrate has been assembled, but not necessarily completely reacted, has provided a good understanding of the process. This protocol describes a gel method for detecting and analyzing yeast splicing complexes formed in vitro on a radiolabeled pre-mRNA substrate. Complexes formed during the splicing reaction are quenched by dilution and addition of an excess of RNA, which is thought to strip nonspecifically bound proteins from the labeled substrate RNA. After loading on a low-percentage, low-cross-linking ratio composite agarose-acrylamide gel (in 10% glycerol), labeled bands are detected. These can be extracted and shown to contain small nuclear RNAs (snRNAs) and partly reacted pre-mRNA.

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

PubMed

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.

Novel microinjector for carrying bone substitutes for bone regeneration in periodontal diseases.

PubMed

Tsai, Hsiao-Cheng; Li, Yi-Chen; Young, Tai-Horng; Chen, Min-Huey

2016-01-01

Traditionally, guide bone regeneration (GBR) was a widely used method for repairing bone lost from periodontal disease. There were some disadvantages associated with the GBR method, such as the need for a stable barrier membrane and a new creative cavity during the surgical process. To address these disadvantages, the purpose of this study was to evaluate a novel microinjector developed for dental applications. The microinjector was designed to carry bone graft substitutes to restore bone defects for bone regeneration in periodontal diseases. The device would be used to replace the GBR method. In this study, the injected force and ejected volume of substitutes (including air, water, and ethanol) were defined by Hooke's law (n = 3). The optimal particle size of bone graft substitutes was determined by measuring the recycle ratio of bone graft substitutes from the microinjector (n = 3). Furthermore, a novel agarose gel model was used to evaluate the feasibility of the microinjector. The current study found that the injected force was less than 0.4 N for obtaining the ejected volume of approximately 2 mL, and when the particle size of tricalcium phosphate (TCP) was smaller than 0.5 mm, 80% TCP could be ejected from the microinjector. Furthermore, by using an agarose model to simulate the periodontal soft tissue, it was also found that bone graft substitutes could be easily injected into the gel. The results confirmed the feasibility of this novel microinjector for dental applications to carry bone graft substitutes for the restoration of bone defects of periodontal disease. Copyright © 2015. Published by Elsevier B.V.

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers.

PubMed

Hanna-Addams, Sarah; Wang, Zhigao

2018-04-26

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Extraction of three-dimensional silver nanostructures with supercritical fluid

NASA Astrophysics Data System (ADS)

Taguchi, Natsuo; Takeyasu, Nobuyuki; Kawata, Satoshi

2018-02-01

In a previous report, a self-growing approach was proposed for fabricating complex silver nanostructures, where silver dendrites were grown at silver nanoseeds in silver ion solution owing to plasmonic heating with ultraviolet light. Structures were deformed or destroyed when they were extracted with acetone and dried in air. In this Letter, we discuss the use of supercritical carbon dioxide fluid for the nondestructive extraction of nanostructures. We show the experimental results and discuss the laser power dependence of resultant structures. Another experiment was performed for nanostructure growth inside an agarose gel as a matrix. Silver nanostructures were immobilized without damage in an agarose skeleton network.

Multichannel microscale system for high throughput preparative separation with comprehensive collection and analysis

DOEpatents

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.

Easy-to-use microfluidic chip for long-term 3D-cell cultures

NASA Astrophysics Data System (ADS)

Bunge, Frank; van den Driesche, Sander; Vellekoop, Michael J.

2017-05-01

We present a microfluidic chip for an easy setup of a 3D-culture of mammalian cells. The chip contains feeding structures and gas supply for long-term cultivation of mammalian cells. The device is fabricated out of hard materials like silicon and glass that are all highly biocompatible. The chip uses the concept of surficial phaseguides that allows the partial filling of a microfluidic chip with liquids based on hydrophobic and hydrophilic surfaces. Here, a suspension of mammalian cells and melted agarose is filled into the chip and is pulled by the capillary pressure on the hydrophilic areas but not on the hydrophobic phaseguides. Consequently, only a part of the chip is filled with the agarose which gels by cooling a form the 3D-cell culture. The unfilled areas are used as supply structures for nutrition and gases. So the supply is based on diffusion and the supply of nutrition and gases is controlled independently. We cultured HaCaT-cells over 24 hours in our device and achieve a good viability.

How-To-Do-It: Recombinant DNA Technology in the High School Biology Laboratory.

ERIC Educational Resources Information Center

Myers, Richard

1988-01-01

Describes a basic biotechnology investigation that includes restriction and ligation of plasmid DNA, transformation of bacteria and cloning of these bacterial cells. Discusses laboratory procedures and another activity in the identification of unknown plasmids by studying agarose gel electrophoresis photographs. (CW)

QUALITY ASSURANCE CONSIDERATIONS FOR USE OF THE FLUORIMAGER SI AND FRAGMENT ANALYSIS SOFTWARE

EPA Science Inventory

The Fluorimager SI (FSI) from Molecular Dynamics is one of several scanning instruments available for the detection of fluorescent emissions associated with DNA samples in a variety of matrices (agarose and polyacrylamide gels, membranes and microplates). In our laboratory, we m...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schindel, Joshua; Muruganandham, Manickam; Pigge, F. Christopher

Purpose: To present a novel marker-flange, addressing source-reconstruction uncertainties due to the artifacts of a titanium intracavitary applicator used for magnetic resonance imaging (MRI)-guided high-dose-rate (HDR) brachytherapy (BT); and to evaluate 7 different MRI marker agents used for interstitial prostate BT and intracavitary gynecologic HDR BT when treatment plans are guided by MRI. Methods and Materials: Seven MRI marker agents were analyzed: saline solution, Conray-60, copper sulfate (CuSO{sub 4}) (1.5 g/L), liquid vitamin E, fish oil, 1% agarose gel (1 g agarose powder per 100 mL distilled water), and a cobalt–chloride complex contrast (C4) (CoCl{sub 2}/glycine = 4:1). A plastic,more » ring-shaped marker-flange was designed and tested on both titanium and plastic applicators. Three separate phantoms were designed to test the marker-flange, interstitial catheters for prostate BT, and intracavitary catheters for gynecologic HDR BT. T1- and T2-weighted MRI were analyzed for all markers in each phantom and quantified as percentages compared with a 3% agarose gel background. The geometric accuracy of the MR signal for the marker-flange was measured using an MRI-CT fusion. Results: The CuSO{sub 4} and C4 markers on T1-weighted MRI and saline on T2-weighted MRI showed the highest signals. The marker-flange showed hyper-signals of >500% with CuSO{sub 4} and C4 on T1-weighted MRI and of >400% with saline on T2-weighted MRI on titanium applicators. On T1-weighted MRI, the MRI signal inaccuracies of marker-flanges were measured <2 mm, regardless of marker agents, and that of CuSO{sub 4} was 0.42 ± 0.14 mm. Conclusion: The use of interstitial/intracavitary markers for MRI-guided prostate/gynecologic BT was observed to be feasible, providing accurate source pathway reconstruction. The novel marker-flange can produce extremely intense, accurate signals, demonstrating its feasibility for gynecologic HDR BT.« less

Characterization of a novel alkaline arylsulfatase from Marinomonas sp. FW-1 and its application in the desulfation of red seaweed agar.

PubMed

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.

Non-radioactive detection of trinucleotide repeat size variability.

PubMed

Tomé, Stéphanie; Nicole, Annie; Gomes-Pereira, Mario; Gourdon, Genevieve

2014-03-06

Many human diseases are associated with the abnormal expansion of unstable trinucleotide repeat sequences. The mechanisms of trinucleotide repeat size mutation have not been fully dissected, and their understanding must be grounded on the detailed analysis of repeat size distributions in human tissues and animal models. Small-pool PCR (SP-PCR) is a robust, highly sensitive and efficient PCR-based approach to assess the levels of repeat size variation, providing both quantitative and qualitative data. The method relies on the amplification of a very low number of DNA molecules, through sucessive dilution of a stock genomic DNA solution. Radioactive Southern blot hybridization is sensitive enough to detect SP-PCR products derived from single template molecules, separated by agarose gel electrophoresis and transferred onto DNA membranes. We describe a variation of the detection method that uses digoxigenin-labelled locked nucleic acid probes. This protocol keeps the sensitivity of the original method, while eliminating the health risks associated with the manipulation of radiolabelled probes, and the burden associated with their regulation, manipulation and waste disposal.

Nucleation of hydroxyapatite by bone sialoprotein.

PubMed Central

Hunter, G K; Goldberg, H A

1993-01-01

Bone sialoprotein (BSP) and osteopontin, the major phosphorylated proteins of mammalian bone, have been proposed to function in the initiation of mineralization. To test this hypothesis, the effects of BSP and osteopontin on hydroxyapatite crystal formation were determined by using a steady-state agarose gel system. At low calcium phosphate concentrations, no accumulation of calcium and phosphate occurred in control gels or gels containing osteopontin. Gels containing BSP at 1-5 micrograms/ml, however, exhibited a visible precipitation band and significantly elevated Ca + PO4 contents. By powder x-ray diffraction, the precipitate formed in the presence of BSP was shown to be hydroxyapatite. These findings suggest that bone sialoprotein may be involved in the nucleation of hydroxyapatite at the mineralization front of bone. Images Fig. 4 PMID:8397409

Using Restriction Mapping to Teach Basic Skills in the Molecular Biology Lab

ERIC Educational Resources Information Center

Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A.

2007-01-01

Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction…

Visualising DNA in Classrooms Using Nile Blue

ERIC Educational Resources Information Center

Milne, Christine; Roche, Scott; McKay, David

2008-01-01

Giving students the opportunity to extract, manipulate and visualise DNA molecules enhances a constructivist approach to learning about modern techniques in biology and biotechnology Visualisation usually requires agarose gel electrophoresis and staining. In this article, we report on an alternative DNA stain, Nile Blue A, that may be used in the…

Zn2+ blocks annealing of complementary single-stranded DNA in a sequence-selective manner

USDA-ARS?s Scientific Manuscript database

A simple low-temperature EDTA-free agarose gel electrophoresis procedure (LTEAGE) coupled with UV-Vis spectrum and fluorescence quenching analyses was developed and the Zn2+-single-stranded (ss) DNA interaction was investigated under near-physiological conditions. It was found that Zn2+ blocked the...

Strategies for the crystallization of viruses: using phase diagrams and gels to produce 3D crystals of Grapevine fanleaf virus.

PubMed

Schellenberger, Pascale; Demangeat, Gérard; Lemaire, Olivier; Ritzenthaler, Christophe; Bergdoll, Marc; Oliéric, Vincent; Sauter, Claude; Lorber, Bernard

2011-05-01

The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly₂₉₇Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7 Å. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 Å by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies. Copyright © 2011 Elsevier Inc. All rights reserved.

Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

2003-11-01

Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

Gel stretch method: a new method to measure constitutive properties of cardiac muscle cells

NASA Technical Reports Server (NTRS)

Zile, M. R.; Cowles, M. K.; Buckley, J. M.; Richardson, K.; Cowles, B. A.; Baicu, C. F.; Cooper G, I. V.; Gharpuray, V.

1998-01-01

Diastolic dysfunction is an important cause of congestive heart failure; however, the basic mechanisms causing diastolic congestive heart failure are not fully understood, especially the role of the cardiac muscle cell, or cardiocyte, in this process. Before the role of the cardiocyte in this pathophysiology can be defined, methods for measuring cardiocyte constitutive properties must be developed and validated. Thus this study was designed to evaluate a new method to characterize cardiocyte constitutive properties, the gel stretch method. Cardiocytes were isolated enzymatically from normal feline hearts and embedded in a 2% agarose gel containing HEPES-Krebs buffer and laminin. This gel was cast in a shape that allowed it to be placed in a stretching device. The ends of the gel were held between a movable roller and fixed plates that acted as mandibles. Distance between the right and left mandibles was increased using a stepper motor system. The force applied to the gel was measured by a force transducer. The resultant cardiocyte strain was determined by imaging the cells with a microscope, capturing the images with a CCD camera, and measuring cardiocyte and sarcomere length changes. Cardiocyte stress was characterized with a finite-element method. These measurements of cardiocyte stress and strain were used to determine cardiocyte stiffness. Two variables affecting cardiocyte stiffness were measured, the passive elastic spring and viscous damping. The passive spring was assessed by increasing the force on the gel at 1 g/min, modeling the resultant stress vs. strain relationship as an exponential [sigma = A/k(ekepsilon - 1)]. In normal cardiocytes, A = 23.0 kN/m2 and k = 16. Viscous damping was assessed by examining the loop area between the stress vs. strain relationship during 1 g/min increases and decreases in force. Normal cardiocytes had a finite loop area = 1.39 kN/m2, indicating the presence of viscous damping. Thus the gel stretch method provided accurate measurements of cardiocyte constitutive properties. These measurements have allowed the first quantitative assessment of passive elastic spring properties and viscous damping in normal mammalian cardiocytes.

Constitutive properties of adult mammalian cardiac muscle cells

NASA Technical Reports Server (NTRS)

Zile, M. R.; Richardson, K.; Cowles, M. K.; Buckley, J. M.; Koide, M.; Cowles, B. A.; Gharpuray, V.; Cooper, G. 4th

1998-01-01

BACKGROUND: The purpose of this study was to determine whether changes in the constitutive properties of the cardiac muscle cell play a causative role in the development of diastolic dysfunction. METHODS AND RESULTS: Cardiocytes from normal and pressure-hypertrophied cats were embedded in an agarose gel, placed on a stretching device, and subjected to a change in stress (sigma), and resultant changes in cell strain (epsilon) were measured. These measurements were used to examine the passive elastic spring, viscous damping, and myofilament activation. The passive elastic spring was assessed in protocol A by increasing the sigma on the agarose gel at a constant rate to define the cardiocyte sigma-versus-epsilon relationship. Viscous damping was assessed in protocol B from the loop area between the cardiocyte sigma-versus-epsilon relationship during an increase and then a decrease in sigma. In both protocols, myofilament activation was minimized by a reduction in [Ca2+]i. Myofilament activation effects were assessed in protocol C by defining cardiocyte sigma versus epsilon during an increase in sigma with physiological [Ca2+]i. In protocol A, the cardiocyte sigma-versus-epsilon relationship was similar in normal and hypertrophied cells. In protocol B, the loop area was greater in hypertrophied than normal cardiocytes. In protocol C, the sigma-versus-epsilon relation in hypertrophied cardiocytes was shifted to the left compared with normal cells. CONCLUSIONS: Changes in viscous damping and myofilament activation in combination may cause pressure-hypertrophied cardiocytes to resist changes in shape during diastole and contribute to diastolic dysfunction.

Benchmarking the ERG valve tip and MRI Interventions Smart Flow neurocatheter convection-enhanced delivery system's performance in a gel model of the brain: employing infusion protocols proposed for gene therapy for Parkinson's disease

NASA Astrophysics Data System (ADS)

Sillay, Karl; Schomberg, Dominic; Hinchman, Angelica; Kumbier, Lauren; Ross, Chris; Kubota, Ken; Brodsky, Ethan; Miranpuri, Gurwattan

2012-04-01

Convection-enhanced delivery (CED) is an advanced infusion technique used to deliver therapeutic agents into the brain. CED has shown promise in recent clinical trials. Independent verification of published parameters is warranted with benchmark testing of published parameters in applicable models such as gel phantoms, ex vivo tissue and in vivo non-human animal models to effectively inform planned and future clinical therapies. In the current study, specific performance characteristics of two CED infusion catheter systems, such as backflow, infusion cloud morphology, volume of distribution (mm3) versus the infused volume (mm3) (Vd/Vi) ratios, rate of infusion (µl min-1) and pressure (mmHg), were examined to ensure published performance standards for the ERG valve-tip (VT) catheter. We tested the hypothesis that the ERG VT catheter with an infusion protocol of a steady 1 µl min-1 functionality is comparable to the newly FDA approved MRI Interventions Smart Flow (SF) catheter with the UCSF infusion protocol in an agarose gel model. In the gel phantom models, no significant difference was found in performance parameters between the VT and SF catheter. We report, for the first time, such benchmark characteristics in CED between these two otherwise similar single-end port VT with stylet and end-port non-stylet infusion systems. Results of the current study in agarose gel models suggest that the performance of the VT catheter is comparable to the SF catheter and warrants further investigation as a tool in the armamentarium of CED techniques for eventual clinical use and application.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

ERIC Educational Resources Information Center

Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

2006-01-01

We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

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

ERIC Educational Resources Information Center

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…

Molecular Identification of Human Fungal Pathogens

DTIC Science & Technology

2009-03-01

initially, had a moderate growth rate, were olivaceous to olivaceous- gray with a black reverse, and became velvety at maturity. Both E. angulospora and Exo...Biolabs, Beverly, MA) at 37°C 158 for 3h and then separated on a 3% NuSieve GTG agarose gel (Cambrex Bio Science Inc., 159 Rockland, ME). Sizes were

Use PCR and a Single Hair To Produce a "DNA Fingerprint."

ERIC Educational Resources Information Center

Campbell, A. Malcolm; And Others

1997-01-01

Presents a laboratory procedure that involves students extracting their own DNA from a single hair follicle, using the polymerase chain reaction (PCR) to amplify a polymorphic locus, performing electrophoresis on the PCR products on an agarose gel, and visualizing the alleles to generate a "DNA fingerprint." Discusses theoretical background,…

Dynamics of a camphoric acid boat at the air-water interface

NASA Astrophysics Data System (ADS)

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).

D-glucose derived novel gemini surfactants: synthesis and study of their surface properties, interaction with DNA, and cytotoxicity.

PubMed

Kumar, Vikash; Chatterjee, Amrita; Kumar, Nupur; Ganguly, Anasuya; Chakraborty, Indranil; Banerjee, Mainak

2014-10-09

Four new D-glucose derived m-s-m type gemini surfactants with variable spacer and tail length have been synthesized by a simple and efficient synthetic methodology utilizing the free C-3 hydroxy group of diisopropylidene glucose. The synthetic route to these gemini surfactants with a quaternary ammonium group as polar head group involves a sequence of simple reactions including alkylation, imine formation, quaternization of amine etc. The surface properties of the new geminis were evaluated by surface tension and conductivity measurements. These gemini surfactants showed low cytotoxicity by MTT assay on HeLa cell line. The DNA binding capabilities of these surfactants were determined by agarose gel electrophoresis, fluorescence titration, and DLS experiments. The preliminary studies by agarose gel electrophoresis indicated chain length dependent DNA binding abilities, further supported by ethidium bromide exclusion experiments. Two of the D-glucose derived gemini surfactants showed effective binding with pET-28a plasmid DNA (pDNA) at relatively low N/P ratio (i.e., cationic nitrogen/DNA phosphate molar ratio). Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular typing of uropathogenic E. coli strains by the ERIC-PCR method.

PubMed

Ardakani, Maryam Afkhami; Ranjbar, Reza

2016-04-01

Escherichia coli (E. coli) is the most common cause of urinary infections in hospitals. The aim of this study was to evaluate the ERIC-PCR method for molecular typing of uropathogenic E. coli strains isolated from hospitalized patients. In a cross sectional study, 98 E. coli samples were collected from urine samples taken from patients admitted to Baqiyatallah Hospital from June 2014 to January 2015. The disk agar diffusion method was used to determine antibiotic sensitivity. DNA proliferation based on repetitive intergenic consensus was used to classify the E. coli strains. The products of proliferation were electrophoresed on 1.5% agarose gel, and their dendrograms were drawn. The data were analyzed by online Insillico software. The method used in this research proliferated numerous bands (4-17 bands), ranging from 100 to 3000 base pairs. The detected strains were classified into six clusters (E1-E6) with 70% similarity between them. In this study, uropathogenic E. coli strains belonged to different genotypic clusters. It was found that ERIC-PCR had good differentiation power for molecular typing of uropathogenic E. coli strains isolated from the patients in the study.

Enhanced Positive-Contrast Visualization of Paramagnetic Contrast Agents Using Phase Images

PubMed Central

Mills, Parker H.; Ahrens, Eric T.

2009-01-01

Iron oxide–based MRI contrast agents are increasingly being used to noninvasively track cells, target molecular epitopes, and monitor gene expression in vivo. Detecting regions of contrast agent accumulation can be challenging if resulting contrast is subtle relative to endogenous tissue hypointensities. A postprocessing method is presented that yields enhanced positive-contrast images from the phase map associated with T2*-weighted MRI data. As examples, the method was applied to an agarose gel phantom doped with superparamagnetic iron-oxide nanoparticles and in vivo and ex vivo mouse brains inoculated with recombinant viruses delivering transgenes that induce overexpression of paramagnetic ferritin. Overall, this approach generates images that exhibit a 1- to 8-fold improvement in contrast-to-noise ratio in regions where paramagnetic agents are present compared to conventional magnitude images. This approach can be used in conjunction with conventional T2* pulse sequences, requires no prescans or increased scan time, and can be applied retrospectively to previously acquired data. PMID:19780169

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Electrophoresis gel image processing and analysis using the KODAK 1D software.

PubMed

Pizzonia, J

2001-06-01

The present article reports on the performance of the KODAK 1D Image Analysis Software for the acquisition of information from electrophoresis experiments and highlights the utility of several mathematical functions for subsequent image processing, analysis, and presentation. Digital images of Coomassie-stained polyacrylamide protein gels containing molecular weight standards and ethidium bromide stained agarose gels containing DNA mass standards are acquired using the KODAK Electrophoresis Documentation and Analysis System 290 (EDAS 290). The KODAK 1D software is used to optimize lane and band identification using features such as isomolecular weight lines. Mathematical functions for mass standard representation are presented, and two methods for estimation of unknown band mass are compared. Given the progressive transition of electrophoresis data acquisition and daily reporting in peer-reviewed journals to digital formats ranging from 8-bit systems such as EDAS 290 to more expensive 16-bit systems, the utility of algorithms such as Gaussian modeling, which can correct geometric aberrations such as clipping due to signal saturation common at lower bit depth levels, is discussed. Finally, image-processing tools that can facilitate image preparation for presentation are demonstrated.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

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

NASA Astrophysics Data System (ADS)

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."

Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

NASA Astrophysics Data System (ADS)

Liu, Ping; Zhao, Liangliang; Wu, Xia; Huang, Fei; Wang, Minqin; Liu, Xiaodan

2014-03-01

It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL-1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)-Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts.

Low-level lasers and mRNA levels of reference genes used in Escherichia coli

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

2016-11-01

Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

Integrating-Sphere Measurements for Determining Optical Properties of Tissue-Engineered Oral Mucosa

NASA Astrophysics Data System (ADS)

Ionescu, A. M.; Cardona, J. C.; Garzón, I.; Oliveira, A. C.; Ghinea, R.; Alaminos, M.; Pérez, M. M.

2015-02-01

Surgical procedures carried out in the oral and maxillofacial region can result in large tissue defects. Accounting for the shortage of oral mucosa to replace the excised tissues, different models of an organotypic substitute of the oral mucosa generated by tissue engineering have recently been proposed. In this work, the propagation of light radiation through artificial human oral mucosa substitutes based on fibrin-agarose scaffolds (fibrin, fibrin-0.1% agarose, fibrin-0.2%agarose) is investigated, and their optical properties are determined using the inverse adding-doubling (IAD) method based on integrating-sphere measurements. Similar values for the absorption and scattering coefficients between the fibrin and fibrin-0.1% agarose bioengineered tissues and the native oral mucosa were found. These results suggest the adequacy of these biomaterials for potential clinical use in human oral mucosa applications. These optical properties represent useful references and data for applications requiring the knowledge of the light transport through this type of tissues, applications used in clinical practice. It also provides a new method of information analysis for the quality control of the development of the artificial nanostructured oral mucosa substitutes and its comparison with native oral mucosa tissues.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Effect of coccolith polysaccharides isolated from the coccolithophorid, Emiliania huxleyi, on calcite crystal formation in in vitro CaCO3 crystallization.

PubMed

Kayano, Keisuke; Saruwatari, Kazuko; Kogure, Toshihiro; Shiraiwa, Yoshihiro

2011-02-01

Marine coccolithophorids (Haptophyceae) produce calcified scales "coccoliths" which are composed of CaCO(3) and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO(3) crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO(3) crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO(3) crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.

Analysis of the response of PVA-GTA Fricke-gel dosimeters with clinical magnetic resonance imaging

NASA Astrophysics Data System (ADS)

Collura, Giorgio; Gallo, Salvatore; Tranchina, Luigi; Abbate, Boris Federico; Bartolotta, Antonio; d'Errico, Francesco; Marrale, Maurizio

2018-01-01

Fricke gel dosimeters produced with a matrix of Poly-vinyl alcohol (PVA) cross-linked with glutaraldehyde (GTA) were analyzed with magnetic resonance imaging (MRI). Previous studies based on spectrophotometry showed valuable dosimetric features of these gels in terms of X-ray sensitivity and diffusion of the ferric ions produced after irradiation. In this study, MRI was performed on the gels at 1.5 T with a clinical scanner in order to optimize the acquisition parameters and obtain high contrast between irradiated and non-irradiated samples. The PVA gels were found to offer good linearity in the range of 0-10 Gy and a stable signal for several hours after irradiation. The sensitivity was about 40% higher compared to gels produced with agarose as gelling agent. The effect of xylenol orange (XO) on the MRI signal was also investigated: gel dosimeters made without XO show higher sensitivity to x-rays than those made with XO. The dosimetric accuracy of the 3D gels was investigated by comparing their MRI response to percentage depth dose and transversal dose profile measurements made with an ionization chamber in a water phantom. The comparison of PVA-GTA gels with and without XO showed that the chelating agent reduces the MRI sensitivity of the gels. Depth-dose and transversal dose profiles acquired by PVA-GTA gels without XO are more accurate and consistent with the ionization chamber data. However, diffusion effects hinder accurate measurements in the steep dose gradient regions and they should be further reduced by modifying the gel matrix and/or by minimizing the delay between irradiation and imaging.

Isolation of "Caenorhabditis elegans" Genomic DNA and Detection of Deletions in the "unc-93" Gene Using PCR

ERIC Educational Resources Information Center

Lissemore, James L.; Lackner, Laura L.; Fedoriw, George D.; De Stasio, Elizabeth A.

2005-01-01

PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the…

S14 as a Therapeutic Target in Breast Cancer

DTIC Science & Technology

2006-08-01

stained agarose gel is shown. Total RNA was analyzed by RT-PCR. Templates (500 ng) were from HepG2 hepatocarcinoma , T47D lipogenic breast cancer, and...cancer, but not MCF10a mammary epithelial or HepG2 hepatocarcinoma cells (not shown). We attempted to reduce S14 mRNA and protein expression using

Agarose Gel Electrophoresis System in the Classroom: Detection of DNA Strand Breaks through the Alteration of Plasmid Topology

ERIC Educational Resources Information Center

De Mattos, J. C. P.; Dantas, F. J. S.; Caldeira-de-Araujo, A.; Moraes, M. O.

2004-01-01

Good quality scientific teaching depends on the ability of researchers to translate laboratory experiments into high school and undergraduate classes, bridging the advanced and basic science with common knowledge. A fast-growing field in biomedical sciences is oxidative stress, which has been associated to several diseases, including cancer and…

Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

PubMed

Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

2011-01-01

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.

An ARMS-based technique for sex determination of red panda (Ailurus fulgens).

PubMed

Li, Yuzhi; Xu, Xiao; Zhang, Liang; Zhang, Zhihe; Shen, Fujun; Zhang, Wenping; Yue, Bisong

2011-03-01

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex. © 2010 Blackwell Publishing Ltd.

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings 1

PubMed Central

Lee, Sung-Chul; West, Charles A.

1981-01-01

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis. PMID:16661728

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; 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 <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and 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.

Use of FTA cards for the storage of breast carcinoma nucleic acid on fine-needle aspiration samples.

PubMed

Peluso, Anna Lucia; Cascone, Anna Maria; Lucchese, Lucrezia; Cozzolino, Immacolata; Ieni, Antonio; Mignogna, Chiara; Pepe, Stefano; Zeppa, Pio

2015-10-01

The preservation and storage of nucleic acids is important for DNA molecular techniques. The material obtained by fine-needle aspiration (FNA) is often scanty and can not be wasted. FTA cards are filter papers that immobilize and stabilize nucleic acids and can be stored at room temperature. The current study evaluated whether nucleic acids of breast carcinoma cells, obtained by FNA in a clinical setting, may be collected, stored, and preserved on FTA cards. Thirty breast carcinoma, 5 non-Hodgkin lymphoma (NHL), and 5 benign reactive lymph node (RLN) cell samples obtained by FNA were stored at -80 °C and on FTA cards. DNA extraction and polymerase chain reaction were performed on cells at -80 °C and on 2 punched disks of FTA cards. Fifty nanograms of extracted DNA from both sample types were used to amplify the Janus Kinase 2 (JAK2) gene. The mean value of DNA extracted from breast carcinoma cells was 28.19 ng/µL for that stored at -80 °C and 3.28 ng/µL for that stored on FTA cards. Agarose gel analysis demonstrated expected bands of DNA in 29 cases (97%) with both methods. The mean value of DNA extracted from NHL and RLN samples was 37.54 ng/µL and 4.28 ng/µL, respectively, and agarose gel analysis demonstrated bands of high molecular weight DNA in both methods. Significant differences in DNA yield were found between storage at -80 °C and FTA cards (P<.0001), but no differences were detected between 260/280 nm ratios in breast carcinoma and NHL/RLN samples. FTA cards can be conveniently used for the storage of breast carcinoma cells obtained by FNA, thus providing a reliable alternative to traditional methods. © 2015 American Cancer Society.

Lipoprotein Agarose Gel Electrophoresis. Application in HDL-Cholesterol Methodology.

DTIC Science & Technology

1985-06-01

on determination of high-density lipo- protein cholesterol by precipitation with sodium phosphotungstate- magnesium. Clin Chem 25(4):560 (1979). 6...determinations of cholesterol levels in supernates obtained after addition of various amounts of precipitants , lipo- protein electrophoresis can help to...plotted against the corresponding volumes of sodium phosphotungstate-MgCl2 (NAPT) precipitant added. 10 ’ ’’ ’i - n

[Fundamental evaluation of apolipoprotein B-48 by chemiluminescence enzyme immunoassay--identification of apolipoprotein B-48 with immunoblotting].

PubMed

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.

Apparent diffusion coefficient measurement in a moving phantom simulating linear respiratory motion.

PubMed

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.

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

USGS Publications Warehouse

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.

New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

PubMed

Shinohara, Masakazu; Ar Rochmah, Mawaddah; Nakanishi, Kenta; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Bouike, Yoshihiro; Nishio, Hisahide

2017-09-07

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. To establish an improved version of the mCOP-PCR screening system without non-specific amplification. DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.

CisSERS: Customizable in silico sequence evaluation for restriction sites

DOE PAGES

Sharpe, Richard M.; Koepke, Tyson; Harper, Artemus; ...

2016-04-12

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Here, data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated tomore » enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERSenable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERSand results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.« less

CisSERS: Customizable in silico sequence evaluation for restriction sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharpe, Richard M.; Koepke, Tyson; Harper, Artemus

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Here, data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated tomore » enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERSenable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERSand results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.« less

Comparison of Genomic Methods for Differentiating Strains of Enterococcus faecium: Assessment Using Clinical Epidemiologic Data

PubMed Central

Savor, Connie; Pfaller, Michael A.; Kruszynski, Julie A.; Hollis, Richard J.; Noskin, Gary A.; Peterson, Lance R.

1998-01-01

Genomic DNA extracted from 45 vancomycin-resistant Enterococcus faecium (VRE) isolates was cleaved with HindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single “ideal” method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection. PMID:9774587

Biofoam II

DOEpatents

Morrison, R.L.

1994-11-01

Biofoam is a rigid, microcellular organic foam made from organic materials derived from natural products and biological organisms. Starting materials include agar, agarose, gelatin, algin, alginates, gellan gum, and microcrystalline cellulose. The organic material is dissolved in a polar solvent, typically water, and the mixture is gelled. The water in the gel pores is replaced at least once with another solvent to reduce the pore size of the final biofoam. The solvent in the gel pores may be replaced several times. After the final replacement of solvent, the gel is frozen and freeze-dried to form a biofoam. Translucent biofoams are formed by selecting a final solvent that forms very small crystals. A variety of crystalline, fibrous, amorphous, or metallic additives may be incorporated into the foam structure to produce lightweight composite materials with enhanced strength and insulating properties. 1 fig.

Biofoam II

DOEpatents

Morrison, Robert L.

1994-01-01

Biofoam is a rigid, microcellular organic foam made from organic materials derived from natural products and biological organisms. Starting materials include agar, agarose, gelatin, algin, alginates, gellan gum, and microcrystalline cellulose. The organic material is dissolved in a polar solvent, typically water, and the mixture is gelled. The water in the gel pores is replaced at least once with another solvent to reduce the pore size of the final biofoam. The solvent in the gel pores may be replaced several times. After the final replacement of solvent, the gel is frozen and freeze-dried to form a biofoam. Translucent biofoams are formed by selecting a final solvent that forms very small crystals. A variety of crystalline, fibrous, amorphous, or metallic additives may be incorporated into the foam structure to produce lightweight composite materials with enhanced strength and insulating properties.

Comparison of six extraction techniques for isolation of DNA from filamentous fungi.

PubMed

van Burik, J A; Schreckhise, R W; White, T C; Bowden, R A; Myerson, D

1998-10-01

Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for cross-contamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5 approximately = to 2 >3 approximately = to 4 approximately = to 6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures.

Drying techniques for the visualisation of agarose-based chromatography media by scanning electron microscopy.

PubMed

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

2017-03-01

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub-micron level. Achieving suitable drying conditions is especially important with agarose-based chromatography resins, as over-drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under-drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect™/d w ≈85 µm and Capto™ Adhere/d w ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose-based chromatography media. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis.

PubMed

De Santis, Riccardo; Ciammaruconi, Andrea; Faggioni, Giovanni; D'Amelio, Raffaele; Marianelli, Cinzia; Lista, Florigio

2009-04-07

Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

Single cell HaloChip assay on paper for point-of-care diagnosis.

PubMed

Ma, Liyuan; Qiao, Yong; Jones, Ross; Singh, Narendra; Su, Ming

2016-11-01

This article describes a paper-based low cost single cell HaloChip assay that can be used to assess drug- and radiation-induced DNA damage at point-of-care. Printing ink on paper effectively blocks fluorescence of paper materials, provides high affinity to charged polyelectrolytes, and prevents penetration of water in paper. After exposure to drug or ionizing radiation, cells are patterned on paper to create discrete and ordered single cell arrays, embedded inside an agarose gel, lysed with alkaline solution to allow damaged DNA fragments to diffuse out of nucleus cores, and form diffusing halos in the gel matrix. After staining DNA with a fluorescent dye, characteristic halos formed around cells, and the level of DNA damage can be quantified by determining sizes of halos and nucleus with an image processing program based on MATLAB. With its low fabrication cost and easy operation, this HaloChip on paper platform will be attractive to rapidly and accurately determine DNA damage for point-of-care evaluation of drug efficacy and radiation condition. Graphical Abstract Single cell HaloChip on paper.

RF HEATING OF MRI-ASSISTED CATHETER STEERING COILS FOR INTERVENTIONAL MRI

PubMed Central

Settecase, Fabio; Hetts, Steven W.; Martin, Alastair J.; Roberts, Timothy P. L.; Bernhardt, Anthony F.; Evans, Lee; Malba, Vincent; Saeed, Maythem; Arenson, Ronald L.; Kucharzyk, Walter; Wilson, Mark W.

2010-01-01

RATIONALE AND OBJECTIVES To assess magnetic resonance imaging (MRI) radiofrequency (RF) related heating of conductive wire coils used in magnetically steerable endovascular catheters. MATERIALS AND METHODS A 3-axis microcoil was fabricated onto a 1.8 Fr catheter tip. In vitro testing was performed in a 1.5 T MRI system using an agarose gel filled vessel phantom, a transmit/receive body RF coil and a steady state free precession (SSFP) pulse sequence, and a fluoroptic thermometry system. Temperature was measured without simulated blood flow at varying distances from magnet isocenter and varying flip angles. Additional experiments were performed with laser-lithographed single-axis microcoil-tipped microcatheters in air and in a saline bath with varied grounding of the microcoil wires. Preliminary in vivo evaluation of RF heating was performed in pigs at 1.5 T with coil-tipped catheters in various positions in the common carotid arteries with SSFP pulse sequence on and off, and under physiologic flow and zero flow conditions. RESULTS In tissue-mimicking agarose gel, RF heating resulted in a maximal temperature increase of 0.35°C after 15 minutes of imaging, 15 cm from magnet isocenter. For a single axis microcoil, maximal temperature increases were 0.73-1.91°C in air and 0.45-0.55°C in saline. In vivo, delayed contrast enhanced MRI revealed no evidence of vascular injury and histopathological sections from the common carotid arteries confirmed the lack of vascular damage. CONCLUSIONS Microcatheter tip microcoils for endovascular catheter steering in MRI experience minimal RF heating under the conditions tested. These data provide the basis for further in vivo testing of this promising technology for endovascular interventional MRI. PMID:21075019

Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography

PubMed Central

Mousavi Hosseini, Kamran; Nasiri, Saleh

2015-01-01

Background: Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. Methods: PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. Results: Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). Results of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml). Conclusion: It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII. PMID:26034723

Induction of biliary cholangiocarcinoma cell apoptosis by 103Pd cholangial radioactive stent gamma-rays.

PubMed

He, Gui-jin; Sun, Dan-dan; Ji, Da-wei; Sui, Dong-ming; Yu, Fa-qiang; Gao, Qin-yi; Dai, Xian-wei; Gao, Hong; Jiang, Tao; Dai, Chao-liu

2008-06-05

In recent years, interventional tumor therapy, involving implantation of intra-cholangial metal stents through percutaneous trans-hepatic punctures, has provided a new method for treating cholangiocarcinoma. (103)Pd cholangial radioactive stents can concentrate high radioactive dosages into the malignant tumors and kill tumor cells effectively, in order to prevent re-stenosis of the lumen caused by a relapsed tumor. The aim of the present study was to investigate the efficacy of gamma-rays released by the (103)Pd biliary duct radioactive stent in treating cholangiocarcinoma via induction of biliary cholangiocarcinoma cell apoptosis. A group of biliary duct cancer cells was collectively treated with a dose of gamma-rays. Cells were then examined by the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl terazolium-bromide (MTT) technique for determining the inhibition rate of the biliary duct cancer cells, as well as with other methods including electron microscopy, DNA agarose gel electrophoresis, and flow cytometry were applied for the evaluation of their morphological and biochemical characteristics. The growth curve and the growth inhibition rate of the cells were determined, and the changes in the ultrastructure of the cholangiocarcinoma cells and the DNA electrophoresis bands were examined under a UV-lamp. The gamma-ray released by (103)Pd inhibited cholangiocarcinoma cell growth, as demonstrated when the growth rate of the cells was stunned by a gamma-ray with a dosage larger than 197.321 MBq. Typical features of cholangiocarcinoma cell apoptosis were observed in the 197.321 MBq dosage group, while cell necrosis was observed when irradiated by a dosage above 245.865 MBq. DNA agarose gel electrophoresis results were different between the 197.321 MBq irradiation dosage group, the 245.865 MBq irradiation dosage group, and the control group. (103)Pd radioactive stents which provide a radioactive dosage of 197.321 MBq are effective in the treatment of cholangiocarcinoma; (103)Pd radioactive stents should be useful for the clinical treatment of cholangiocarcinoma.

A PCR method based on 18S rRNA gene for detection of malaria parasite in Balochistan.

PubMed

Shahwani, Zubeda; Aleem, Abdul; Ahmed, Nazeer; Mushtaq, Muhammad; Afridi, Sarwat

2016-12-01

To establish a polymerase chain reaction method based on 18S ribosomal ribonucleic acid gene for the detection of plasmodium deoxyribonucleic acid in patients suffering from malaria symptoms. This cross-sectional study was conducted from September 2013 to October 2014 in district Quetta of Pakistan's Balochistan province. Blood samples were collected from patients suffering from general symptoms of malaria. A polymerase chain reaction-based technique was applied for the diagnosis of malaria and detection of responsible species in the patients who were suspected to carry the parasite. Performance of this polymerase chain reaction method was compared against the microscopy results. Parasite number was also calculated for microscopy positive samples.All samples after the genomic deoxyribonucleic acid isolation were subjected to polymerase chain reaction amplification and agarose gel electrophoresis. Of the 200 samples, 114(57%) were confirmed as positive and 86(43%) as negative for malaria by microscopy. Polymerase chain reaction identified 124(62%) samples as positive and 76(38%) as negative for malaria. The comparative analysis of both diagnostic methods confirmed 109(54.5%) samples as positive by both techniques. Besides, 5(6.58%) samples were identified as false positive and 15(12.1%) samples as false negative by polymerase chain reaction. Sensitivity, specificity and positive predictive values for polymerase chain reaction in comparison to microscopy were 87.98%, 93.42% and 96%, respectively. Polymerase chain reaction-based methods in malaria diagnosis and species identification were found to be more effective than other techniques.

Iron-Doped (La,Sr)MnO3 Manganites as Promising Mediators of Self-Controlled Magnetic Nanohyperthermia.

PubMed

Shlapa, Yulia; Kulyk, Mykola; Kalita, Viktor; Polek, Taras; Tovstolytkin, Alexandr; Greneche, Jean-Marc; Solopan, Sergii; Belous, Anatolii

2016-12-01

Fe-doped La0.77Sr0.23Mn1 - y Fe y O3 nanoparticles have been synthesized by sol-gel method, and ceramic samples based on them were sintered at 1613 K. Crystallographic and magnetic properties of obtained nanoparticles and ceramic samples have been studied. It has been established that cell volume for nanoparticles increases with growing of iron content, while this dependence displays an opposite trend in the case of ceramic samples. Mössbauer investigations have shown that in all samples, the oxidation state of iron is +3. According to magnetic studies, at room temperature, both nanoparticles and ceramic samples with y ≤ 0.06 display superparamagnetic properties and samples with y ≥ 0.08 are paramagnetic. Magnetic fluids based on La0.77Sr0.23Mn1 - y Fe y O3 nanoparticles and aqua solution of agarose have been prepared. It has been established that heating efficiency of nanoparticles under an alternating magnetic field decreases with growing of iron content.

Investigation of irradiated rats DNA in the presence of Cu(II) chelates of amino acids Schiff bases.

PubMed

Karapetyan, N H; Torosyan, A L; Malakyan, M; Bajinyan, S A; Haroutiunian, S G

2016-01-01

The new synthesized Cu(II) chelates of amino acids Schiff bases were studied as a potential radioprotectors. Male albino rats of Wistar strain were exposed to X-ray whole-body irradiation at 4.8 Gy. This dose caused 30% mortality of the animals (LD30). The survival of animals exposed to radiation after preliminary administration of 10 mg/kg Cu(II)(Nicotinyl-L-Tyrosinate)2 or Cu(II)(Nicotinyl-L-Tryptophanate)2 prior to irradiation was registered about 80 and 100% correspondingly. Using spectrophotometric melting and agarose gel electrophoresis methods, the differences between the DNA isolated from irradiated rats and rats pretreated with Cu(II) chelates were studied. The fragments of DNA with different breaks were revealed in DNA samples isolated from irradiated animals. While, the repair of the DNA structure was observed for animals pretreated with the Cu(II) chelates. The results suggested that pretreatment of the irradiated rats with Cu(II)(Nicotinyl-L-Tyrosinate)2 and Cu(II)(Nicotinyl-L-Tryptophanate)2 compounds improves the liver DNA characteristics.

Stabilization of the Inverse Laplace Transform of Multiexponential Decay through Introduction of a Second Dimension

PubMed Central

Celik, Hasan; Bouhrara, Mustapha; Reiter, David A.; Fishbein, Kenneth W.; Spencer, Richard G.

2013-01-01

We propose a new approach to stabilizing the inverse Laplace transform of a multiexponential decay signal, a classically ill-posed problem, in the context of nuclear magnetic resonance relaxometry. The method is based on extension to a second, indirectly detected, dimension, that is, use of the established framework of two-dimensional relaxometry, followed by projection onto the desired axis. Numerical results for signals comprised of discrete T1 and T2 relaxation components and experiments performed on agarose gel phantoms are presented. We find markedly improved accuracy, and stability with respect to noise, as well as insensitivity to regularization in quantifying underlying relaxation components through use of the two-dimensional as compared to the one-dimensional inverse Laplace transform. This improvement is demonstrated separately for two different inversion algorithms, nonnegative least squares and non-linear least squares, to indicate the generalizability of this approach. These results may have wide applicability in approaches to the Fredholm integral equation of the first kind. PMID:24035004

Classification of Normal and Male-Sterile Cytoplasms in Maize. II. Electrophoretic Analysis of DNA Species in Mitochondria

PubMed Central

Kemble, R. J.; Gunn, R. E.; Flavell, R. B.

1980-01-01

Mitochondrial DNA preparations were made from 31 maize lines carrying different sources of cytoplasm in the same nuclear genetic background. The DNAs were analyzed by agarose gel electrophoresis. A number of discrete low molecular weight bands were present in all lines. However, only four different DNA banding patterns were observed. These were correlated with the N, T, S and C cytoplasms defined by nuclear fertility restorer genes. Of the 31 cytoplasmic sources examined, six possessed DNA species characteristic of N cytoplasms, four possessed DNA species characteristic of T cytoplasm, 19 possessed DNA species characteristic of S cytoplasm and two possessed DNA species characteristic of C cytoplasm. This classification is in complete agreement with that based on mitochondrial translation products reported in the accompanying paper. No within-group heterogeneity was observed in the DNA banding patterns, indicating a lack of cytoplasmic variation within the four cytoplasmic groups. Attributes of the various methods available for classifying maize cytoplasms are compared and discussed. PMID:17249046

Application of Faecalibacterium 16S rDNA genetic marker for accurate identification of duck faeces.

PubMed

Sun, Da; Duan, Chuanren; Shang, Yaning; Ma, Yunxia; Tan, Lili; Zhai, Jun; Gao, Xu; Guo, Jingsong; Wang, Guixue

2016-04-01

The aim of this study was to judge the legal duty of pollution liabilities by assessing a duck faeces-specific marker, which can exclude distractions of residual bacteria from earlier contamination accidents. With the gene sequencing technology and bioinformatics method, we completed the comparative analysis of Faecalibacterium sequences, which were associated with ducks and other animal species, and found the sequences unique to duck faeces. Polymerase chain reaction (PCR) and agarose gel electrophoresis techniques were used to verify the reliability of both human and duck faeces-specific primers. The duck faeces-specific primers generated an amplicon of 141 bp from 43.3 % of duck faecal samples, 0 % of control samples and 100 % of sewage wastewater samples that contained duck faeces. We present here the initial evidence of Faecalibacterium-based applicability as human faeces-specificity in China. Meanwhile, this study represents the initial report of a Faecalibacterium marker for duck faeces and suggests an independent or supplementary environmental biotechnology of microbial source tracking (MST).

The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

PubMed

Chen, M H; Kuo, S T; Renault, T; Chang, P H

2014-02-01

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus. Copyright © 2013 Elsevier B.V. All rights reserved.

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

PubMed

García-Mena, Jaime; Cano-Ramirez, Claudia; Garibay-Orijel, Claudio; Ramirez-Canseco, Sergio; Poggi-Varaldo, Héctor M

2005-06-01

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

PubMed

Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

2016-10-01

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

PubMed

Oza, Mihir D; Prasad, Kamalesh; Siddhanta, A K

2012-08-01

New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications. Copyright © 2012. Published by Elsevier Ltd.

Pulsed-field gel electrophoresis (PFGE): application in population structure studies of bovine mastitis-causing streptococci.

PubMed

Santos-Sanches, Ilda; Chambel, Lélia; Tenreiro, Rogério

2015-01-01

Pulsed-field gel electrophoresis (PFGE) separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens and is a valuable typing scheme to detect and differentiate strains. Particularly, the contour-clamped homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of many bacterial pathogens. Here we describe a PFGE protocol that was applicable to the study of bovine streptococci, namely, Streptococcus agalactiae (group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (group C Streptococcus, GCS), and Streptococcus uberis-which are relevant pathogens causing mastitis, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production.

Maximizing PTH Anabolic Osteoporosis Therapy

DTIC Science & Technology

2015-09-01

SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18 . NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c...normalization or endogenous controls and calculates fold 263 changes with P values. Gene expression data were normalized to five endogenous controls ( 18S ...adapters were ligated and the sample was size-fractionated (200-300 bp) on an agarose gel. After a final 294 PCR amplification step ( 18 cycles), the

Purification and identification of the fusicoccin binding protein from oat root plasma membrane

NASA Technical Reports Server (NTRS)

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.

Quantification of fetal and total circulatory DNA in maternal plasma samples before and after size fractionation by agarose gel electrophoresis.

PubMed

Hromadnikova, I; Zejskova, L; Doucha, J; Codl, D

2006-11-01

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100-300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300-500 bp fraction. Circulatory DNA extracted from the 100-300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300-500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.

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

PubMed Central

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

Detection of processed genetically modified food using CIM monolithic columns for DNA isolation.

PubMed

Jerman, Sergej; Podgornik, Ales; Cankar, Katarina; Cadet, Neza; Skrt, Mihaela; Zel, Jana; Raspor, Peter

2005-02-11

The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pretreated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix-food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods.

A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

PubMed

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.

A PCR Detection Method for Rapid Identification of Melissococcus pluton in Honeybee Larvae

PubMed Central

Govan, V. A.; Brözel, V.; Allsopp, M. H.; Davison, S.

1998-01-01

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. PMID:9572987

Highly parallel single-molecule amplification approach based on agarose droplet polymerase chain reaction for efficient and cost-effective aptamer selection.

PubMed

Zhang, Wei Yun; Zhang, Wenhua; Liu, Zhiyuan; Li, Cong; Zhu, Zhi; Yang, Chaoyong James

2012-01-03

We have developed a novel method for efficiently screening affinity ligands (aptamers) from a complex single-stranded DNA (ssDNA) library by employing single-molecule emulsion polymerase chain reaction (PCR) based on the agarose droplet microfluidic technology. In a typical systematic evolution of ligands by exponential enrichment (SELEX) process, the enriched library is sequenced first, and tens to hundreds of aptamer candidates are analyzed via a bioinformatic approach. Possible candidates are then chemically synthesized, and their binding affinities are measured individually. Such a process is time-consuming, labor-intensive, inefficient, and expensive. To address these problems, we have developed a highly efficient single-molecule approach for aptamer screening using our agarose droplet microfluidic technology. Statistically diluted ssDNA of the pre-enriched library evolved through conventional SELEX against cancer biomarker Shp2 protein was encapsulated into individual uniform agarose droplets for droplet PCR to generate clonal agarose beads. The binding capacity of amplified ssDNA from each clonal bead was then screened via high-throughput fluorescence cytometry. DNA clones with high binding capacity and low K(d) were chosen as the aptamer and can be directly used for downstream biomedical applications. We have identified an ssDNA aptamer that selectively recognizes Shp2 with a K(d) of 24.9 nM. Compared to a conventional sequencing-chemical synthesis-screening work flow, our approach avoids large-scale DNA sequencing and expensive, time-consuming DNA synthesis of large populations of DNA candidates. The agarose droplet microfluidic approach is thus highly efficient and cost-effective for molecular evolution approaches and will find wide application in molecular evolution technologies, including mRNA display, phage display, and so on. © 2011 American Chemical Society

Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

NASA Astrophysics Data System (ADS)

Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

1994-07-01

We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

Further Development of Scaffolds for Regeneration of Nerves

NASA Technical Reports Server (NTRS)

Sakamoto, Jeffrey; Tuszynski, Mark

2009-01-01

Progress has been made in continuing research on scaffolds for the guided growth of nerves to replace damaged ones. The scaffolds contain pores that are approximately cylindrical and parallel, with nearly uniform widths ranging from tens to hundreds of microns. At the earlier stage of development, experimental scaffolds had been made from agarose hydrogel. Such a scaffold was made in a multistep process in which poly(methyl methacrylate) [PMMA] fibers were used as templates for the pores. The process included placement of a bundle of the PMMA fibers in a tube, filling the interstices in the tube with a hot agarose solution, cooling to turn the solution into a gel, and then immersion in acetone to dissolve the PMMA fibers. The scaffolds were typically limited to about 25 pores per scaffold, square cross sections of no more than about 1.5 by 1.5 mm, and lengths of no more than about 2 mm.

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

NASA Astrophysics Data System (ADS)

Akihiro Hattori,; Kenji Yasuda,

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.

Millimeter-scale alkalinity measurement in marine sediment using DET probes and colorimetric determination.

PubMed

Metzger, E; Viollier, E; Simonucci, C; Prévot, F; Langlet, D; Jézéquel, D

2013-10-01

Constrained DET (Diffusive Equilibration in Thin films) probes equipped with 75 sampling layers of agarose gel (DGT Research(©)) were used to sample bottom and pore waters in marine sediment with a 2 mm vertical resolution. After retrieval, each piece of hydrogel, corresponding to 25 μL, was introduced into 1 mL of colorimetric reagent (CR) solution consisting of formic acid and bromophenol blue. After the elution/reaction time, absorbance of the latter mixture was read at 590 nm and compared to a calibration curve obtained with the same protocol applied to mini DET probes soaked in sodium hydrogen carbonate standard solutions. This method allows rapid alkalinity determinations for the small volumes of anoxic pore water entrapped into the gel. The method was assessed on organic-rich coastal marine sediments from Thau lagoon (France). Alkalinity values in the overlying waters were in agreement with data obtained by classical sampling techniques. Pore water data showed a progressive increase of alkalinity in the sediment from 2 to 10 mmol kg(-1), corresponding to anaerobic respiration in organic-rich sediments. Moreover, replicates of high-resolution DET profiles showed important lateral heterogeneity at a decimeter scale. This underlines the importance of high-resolution spatial methods for alkalinity profiling in coastal marine systems. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.

PubMed

Weiss, Agnes; Jérôme, Valérie; Freitag, Ruth

2007-06-15

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.

Molecular strain typing of Brucella abortus isolates from Italy by two VNTR allele sizing technologies.

PubMed

De Santis, Riccardo; Ancora, Massimo; De Massis, Fabrizio; Ciammaruconi, Andrea; Zilli, Katiuscia; Di Giannatale, Elisabetta; Pittiglio, Valentina; Fillo, Silvia; Lista, Florigio

2013-10-01

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

PubMed Central

Hashemipetroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

2018-01-01

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species PMID:29443017

Genetic diversity analysis of Chrysopidae family (Insecta, Neuroptera) via molecular markers.

PubMed

Yari, Kheirollah; Mirmoayedi, Alinaghi; Marami, Marzieh; Kazemi, Elham; Kahrizi, Danial

2014-09-01

In entomology, improvement of molecular methods would be beneficial tools for accurate identification and detecting the genetic diversity of insect species to discover a corroborative evidence for the traditional classification based on morphology. The aim of this study was focused on RAPD-PCR method for distinguishing the genetic diversity between eight species of Chrysopidae family. In current research, many specimens were collected in different locations of Tehran province (Iran), between them 24 specimens were identified. The wing venation, male genitalia and other morphological characters were used for identification and also the sexing of species was recognized with study of external genitalia. Then, the DNA was extracted with CTAB method. The RAPD-PCR method was carried out with twenty random primers. The agarose gel electrophoresis was used for separation of the PCR products. Based on electrophoresis results, 133 bands were amplified and between them, 126 bands were poly-morph and others were mono-morph. Also, among the applied primers, the primers OPA02 with 19 bands and OPA03 with 8 bands were amplified the maximum and minimum of bands, respectively. The results showed that 80.35 and 73.21 % of genetic similarity existed between Chrysopa pallens-Chrysopa dubitans, and between the Chrysoperla kolthoffi and Chrysoperla carnea, respectively. The minimum (45.53 %) of genetic similarity was observed between C. kolthoffi and C. dubitans, and the maximum (0.80 %) was seen between C. pallens and C. dubitans.

Molecular Epidemiology of Mycobacterium tuberculosis Isolates in 100 Patients With Tuberculosis Using Pulsed Field Gel Electrophoresis

PubMed Central

Pooideh, Mohammad; Jabbarzadeh, Ismail; Ranjbar, Reza; Saifi, Mahnaz

2015-01-01

Background: Tuberculosis (TB) is a widespread infectious disease. Today, TB has created a public health crisis in the world. Genotyping of Mycobacterium tuberculosis isolates is useful for surveying the dynamics of TB infection, identifying new outbreaks, and preventing the disease. Different molecular methods for clustering of M. tuberculosis isolates have been used. Objectives: During a one year study of genotyping, 100 M. tuberculosis isolates from patients referred to Pasteur Institute of Iran were collected and their genotyping was accomplished using pulsed field gel electrophoresis (PFGE) method. Materials and Methods: Identification of all M. tuberculosis isolates was accomplished using standard biochemical and species-specific polymerase chain reaction (PCR) methods. Antibiotic susceptibility tests were performed using proportional method. After preparing PFGE plaques for each isolate of M. tuberculosis, XbaI restriction enzyme was applied for genome digestion. Finally, the digested DNA fragments were separated on 1% agarose gel and analyzed with GelCompar II software. Results: Genotyping of the studied isolates in comparison with the molecular weight marker revealed two common types; pulsotype A with 71 isolates and one multidrug resistant mycobacterium (MDR) case, and pulsotype B including 29 isolates and three MDR cases. No correlation between the antibiotypes and pulsotypes was observed. Conclusions: Molecular epidemiology studies of infectious diseases have been useful when bacterial isolates have been clustered in a period of time and in different geographical regions with variable antibiotic resistance patterns. In spite of high geographical differences and different antibiotic resistant patterns, low genetic diversity among the studied TB isolates may refer to the low rate of mutations in XbaI restriction sites in the mycobacterial genome. We also identified three MDR isolates in low-incidence pulsotype B, which could be disseminated and is highly important to consider in TB surveillance programs to prevent the spread of MDR-TB isolates in the population. PMID:26396714

Evaluation of anti-freeze viscosity modifier for potential external tank applications

NASA Technical Reports Server (NTRS)

Lynn, R. O. L.

1981-01-01

Viscosity modifiers and gelling agents were evaluated in combination with ethylene glycol and dimethyl sulfoxide water eutectics. Pectin and agarose are found to gel these eutectics effectively in low concentration, but the anti-freeze protection afforded by these compositions is found to be marginal in simulations of the intended applications. Oxygen vent shutters and vertical metallic surfaces were simulated, with water supplied as a spray, dropwise, and by condensation from the air.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿†

PubMed Central

Pourcel, Christine; Minandri, Fabrizia; Hauck, Yolande; D'Arezzo, Silvia; Imperi, Francesco; Vergnaud, Gilles; Visca, Paolo

2011-01-01

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/. PMID:21147956

A noda-like virus isolated from the sweetpotato pest spodoptera eridania (Cramer) (Lep.; noctuidae)

PubMed

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.

The influence of ionic strength on DNA diffusion in gel networks

NASA Astrophysics Data System (ADS)

Fu, Yuanxi; Jee, Ah-Young; Kim, Hyeong-Ju; Granick, Steve

Cations are known to reduce the rigidity of the DNA molecules by screening the negative charge along the sugar phosphate backbone. This was established by optical tweezer pulling experiment of immobilized DNA strands. However, little is known regarding the influence of ions on the motion of DNA molecules as they thread through network meshes. We imaged in real time the Brownian diffusion of fluorescent labeled lambda-DNA in an agarose gel network in the presence of salt with monovalent or multivalent cations. Each movie was analyzed using home-written program to yield a trajectory of center of the mass and the accompanying history of the shape fluctuations. One preliminary finding is that ionic strength has a profound influence on the slope of the trace of mean square displacement (MSD) versus time. The influence of ionic strength on DNA diffusion in gel networks.

Use of Competitive PCR to Detect and Quantify Haplosporidium nelsoni Infection (MSX disease) in the Eastern Oyster (Crassostrea virginica).

PubMed

Day, J Michael; Franklin, Dean E.; Brown, Bonnie L.

2000-09-01

This study was undertaken to develop a quantitative polymerase chain reaction assay that would improve the utility of PCR for detecting Haplosporidium nelsoni (MSX), a serious parasite of the eastern oyster Crassostrea virginica. A competitive PCR sequence was generated from the H. nelsoni small subunit ribosomal DNA fragment, originally described by Stokes and colleagues, that was amplified by the same PCR primers and had similar amplification performance. Assays performed using competitor dilutions ranging from 0.05 to 500 pg/µl DNA were used to test oyster samples designated using histological techniques as having "light" or "heavy" MSX infections. Visual diagnoses were confirmed equally well with three methods: densitometry of ethidium-bromide-stained agarose, densitometry of SYBRGreen-stained polyacrylamide gels, and analysis by GeneScan 3.0 of fluorescent products detected in ultrathin gels. Oysters diagnosed as negative for MSX tested as negative or light by PCR. Oysters with light MSX infections generally had less than 5 pg/µl infectious DNA. Oysters with heavy infections generally corresponded to 5 pg/µl or greater competitor dilutions.

Core-shell magnetite-silica composite nanoparticles enhancing DNA damage induced by a photoactive platinum-diimine complex in red light.

PubMed

Zhang, Zhigang; Chai, Aiyun

2012-12-01

Lack of solubility under physiological conditions poses an additional risk for toxicity and side effects for intravenous delivery of the photodynamic therapeutic agent in vivo. Employing magnetite-silica composite nanoparticles as carriers of the photodynamic therapeutic agents may be a promising way to solve the problem. In this study, core-shell magnetite-silica composite nanoparticles were prepared by a sol-gel method, and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering, then they were used as carriers of a photoactive platinum diimine complex. The interactions of the photosensitizer-loaded magnetic composite nanoparticles with DNA in red light were monitored by agarose-gel electrophoresis. The results suggest that high doses of magnetite-silica composite nanoparticles might facilitate the transformation of covalently closed circular (ccc)-DNA band to open circular (oc)-DNA band though they are harmless to DNA at their low concentrations, therefore enhancing the extent of DNA damage caused by the metal complex in red light. Copyright © 2012 Elsevier Inc. All rights reserved.

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

PubMed

Hernández, Marta; Rodríguez-Lázaro, David; Zhang, David; Esteve, Teresa; Pla, Maria; Prat, Salomé

2005-05-04

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

Metal (II) Complexes Derived from Naphthofuran-2-carbohydrazide and Diacetylmonoxime Schiff Base: Synthesis, Spectroscopic, Electrochemical, and Biological Investigation

PubMed Central

Sumathi, R. B.; Halli, M. B.

2014-01-01

A new Schiff base and a new series of Co(II), Ni(II), Cu(II), Cd(II), and Hg(II) complexes were synthesized by the condensation of naphthofuran-2-carbohydrazide and diacetylmonoxime. Metal complexes of the Schiff base were prepared from their chloride salts of Co(II), Ni(II), Cu(II), Cd(II), and Hg(II) in ethanol. The ligand along with its metal complexes have been characterized on the basis of analytical data, IR, electronic, mass, 1HNMR, ESR spectral data, thermal studies, magnetic susceptibility, and molar conductance measurements. The nonelectrolytic behaviour of the complexes was assessed from the measured low conductance data. The elemental analysis of the complexes confirm the stoichiometry of the type CuL2Cl2 and MLCl2 where M = Ni(II), Co(II), Cd(II), and Hg(II) and L = Schiff base. The redox property of the Cu(II) complex was investigated by electrochemical method using cyclic voltammetry. In the light of these results, Co(II), Ni(II), and Cu(II) complexes are assigned octahedral geometry, Cd(II), and Hg(II) complexes tetrahedral geometry. In order to evaluate the effect of metal ions upon chelation, both the ligand and its metal complexes were screened for their antibacterial and antifungal activities by minimum inhibitory concentration (MIC) method. The DNA cleaving capacity of all the complexes was analysed by agarose gel electrophoresis method. PMID:24592203

Histidinoalanine, a naturally occurring cross-link derived from phosphoserine and histidine residues in mineral-binding phosphoproteins.

PubMed

Marsh, M E

1986-05-06

Native mineral-containing phosphoprotein particles were isolated from the Heterodont bivalve Macrocallista nimbosa. The native particles are discrete structures about 40 nm in diameter which migrate as a single band during electrophoresis in agarose gels. Removal of the mineral component with ethylenediaminetetraacetic acid dissociates the native protein into nonidentical subunits. The lower molecular weight subunits, representing 8% of the total protein, were obtained by differential centrifugation. The native protein is characterized by a high content of aspartic acid, phosphoserine, phosphothreonine, histidine, and the bifunctional cross-linking residue histidinoalanine. The low molecular weight subunits have the same amino acid composition except for a reduction in histidinoalanine and a corresponding increase in phosphoserine and histidine residues, demonstrating that the alanine portion of the cross-link is derived from phosphoserine residues. Ion-exchange chromatography and molecular sieve chromatography show that the low molecular weight subunits have a similar charge density but differ in molecular weight, and the relative mobilities of the subunits on agarose gels indicate that they are polymers of a single phosphoprotein molecule. The minimum molecular weight of the monomer is about 140 000 on the basis of the amino acid composition. The high molecular weight subunits are rich in histidinoalanine and too large to be resolved by either molecular sieve chromatography or gel electrophoresis. On the basis of the ultrastructural, electrophoretic, chromatographic, and compositional evidence, native phosphoprotein particles are composed of subunits ionically cross-linked via divalent cations. These subunits are variable molecular weight aggregates of a single phosphoprotein molecule covalently cross-linked via histidinoalanine residues. Evidence for a nonenzymatic cross-linking mechanism is discussed.

Agarose encapsulated mesoporous carbonated hydroxyapatite nanocomposites powder for drug delivery.

PubMed

Kolanthai, Elayaraja; Abinaya Sindu, P; Thanigai Arul, K; Sarath Chandra, V; Manikandan, E; Narayana Kalkura, S

2017-01-01

The powder composites are predominantly used for filling of voids in bone and as drug delivery carrier to prevent the infection or inflammatory reaction in the damaged tissues. The objective of this work was to study the synthesis of agarose encapsulation on carbonated hydroxyapatite powder and their biological and drug delivery properties. Mesoporous, nanosized carbonated hydroxyapatite/agarose (CHAp/agarose) powder composites were prepared by solvothermal method and subsequently calcined to study the physico-chemical changes, if it subjected to thermal exposure. The phase of the as-synthesized powder was CHAp/agarose whereas the calcinated samples were non-stoichiometric HAp. The CHAp/agarose nanorods were of length 10-80nm and width 40-190nm for the samples synthesized at temperatures 120°C (ST120) and 150°C (ST150). The calcination process produced spheres (10-50nm) and rods with reduced size (40-120nm length and 20-30nm width). Composites were partially dissolved in SBF solution followed by exhibited better bioactivity than non-stoichiometric HAp confirmed by gravimetric method. Hemo and biocompatibility remained unaffected by presence of agarose or carbonate in the HAp. Specific surface area of the composites was high and exhibited an enhanced amoxicillin and 5-fluorouracil release than the calcined samples. The composites demonstrated a strong antimicrobial activity against E. coli, S. aureus and S. epidermidis. The ST120 showed prolonged drug (AMX and 5-Fcil) release and antimicrobial efficacy than ST150 and calcined samples. This technique would be simple and rapid for composites preparation, to produce high quality crystalline, resorbable, mesoporous and bioactive nanocomposite (CHAp/agarose) powders. This work provides new insight into the role of agarose coated on bioceramics by solvothermal technique and suggests that CHAp/agarose composites powders are promising materials for filling of void in bone and drug delivery applications. Copyright © 2016 Elsevier B.V. All rights reserved.

X- and Y-chromosome-specific variants of the amelogenin gene allow non-invasive sex diagnosis for the detection of pseudohermaphrodite goats.

PubMed

Fábián, Renáta; Kovács, András; Stéger, Viktor; Frank, Krisztián; Egerszegi, István; Oláh, János; Bodó, Szilárd

2017-12-01

The Polled Intersex Syndrome (PIS) is responsible for the absence of horns in homozygous and heterozygous goats causing a female-to-male sex reversal in the homozygous polled genotypic female (XX) goats. A simple and efficient non-invasive method was elaborated to detect the genotypic sex from hair and faecal samples using a pair of primers to amplify the X- and Y-linked alleles of the amelogenin gene. The PCR products were easily distinguishable using agarose gel electrophoresis: we detected an X-specific single band in samples originating from healthy phenotypic females and double (X- and Y-) bands in samples from males. The new PCR method is applicable for diagnosing the sex of PIS-affected animals already as newborn kids, in contrast with the phenotypic findings appearing only after puberty, and thus it may replace the cumbersome chromosome investigations.

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

NASA Astrophysics Data System (ADS)

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

An RNA isolation system for plant tissues rich in secondary metabolites

PubMed Central

2011-01-01

Background Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression. Findings An RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel. Conclusions The present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization. PMID:21443767

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

PubMed

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.

Comparison of five techniques for the detection of Renibacterium salmoninarum in adult coho salmon.

USGS Publications Warehouse

Pascho, R.J.; Elliott, D.G.; Mallett, R.W.; Mulcahy, D.

1987-01-01

Samples of kidney, spleen, coelomic fluid, and blood from 56 sexually mature coho salmon Oncorhynchus kisutch were examined for infection by Renibacterium salmoninarum by five methods. The overall prevalence (all sample types combined) of R. salmoninarum in the fish was 100% by the enzyme-linked immunosorbent assay, 86% by the combined results of the direct fluorescent antibody and the direct filtration-fluorescent antibody techniques, 39% by culture, 11% by counterimmunoelectrophoresis, and 5% by agarose gel immunodiffusion. There was a significant positive correlation (P < 0.001) between the enzyme-linked immunosorbent assay absorbance levels and the counts by fluorescent antibody techniques for kidney, spleen, and coelomic fluid, and significant positive correlations (P < 0.001) in enzyme-linked immunosorbent assay absorbance levels for all four of the sample types.

Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon.

PubMed

Zheng, Lu; Gao, Naiyun; Deng, Yang

2012-01-01

It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples.

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

PubMed

Li, D; Kong, Y; Yu, H; Lehtinen, A; Huang, H; Shen, F; Min, L; Zhou, J; Tang, G; Wang, Q

2008-04-01

A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

[Diffusion of fluorescent and magnetic molecular probes in brain interstitial space].

PubMed

Li, Huai-ye; Zhao, Yue; Zuo, Long; Fu, Yu; Li, Nan; Yuan, Lan; Zhang, Shu-jia; Han, Hong-bin

2015-08-18

To compare the diffusion properties of fluorescent probes dextran-tetramethylrhodamine (DT) and lucifer yellow CH (LY) and magnetic probe gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) in porous media and to screen out a suitable fluorescent probe for optical imaging of brain interstitial space (ISS). Agarose gels sample were divided into DT group, LY group and Gd-DTPA group, and the corresponding molecular probes were imported in each group. The dynamic diffusions of DT and LY in agarose gels at different time points (15, 30, 45, 60, 90, and 120 min) were scanned with laser scanning confocal microscope, the dynamic diffusion of Gd-DTPA was imaged with magnetic resonance imaging. The average diffusion speed of LY were demonstrated to be consistent with those of Gd-DTPA. The LY was introduced into caudate putamen of 18 rats, respectively, the diffusion of LY in the sequential slices of rat brain at different time points (0.5, 1, 2, 3, 7, 11 h) were scanned, and the results were compared with those of rats' brain with Gd-DTPA imported and imaged in vivo with magnetic resonance imaging. The diffusions of the three probes were isotropic in the agarose gels, and the average diffusion speeds of DT, LY and Gd-DTPA were: (0.07±0.02)×10(-2) mm2/s, (1.54±0.47)×10(-2) mm2/s, (1.45±0.50)×10(-2) mm2/s, respectively. The speed of DT was more slower than both LY and Gd-DTPA (ANOVA, F=367.15, P<0.001; Post-Hoc LSD, P<0.001), and there was no significant difference between the speeds of LY and Gd-DTPA (Post-Hoc LSD, P=0.091). The variation tendency of diffusion area of DT was different with both that of LY and that of Gd-DTPA (Bonferroni correction, α=0.0125, P<0.001), and there was no significant difference between LY and Gd-DTPA (Bonferroni correction, α=0.0125, P=0.203), in analysis by repeated measures data of ANOVA. The diffusions of LY and Gd-DTPA were anisotropy in rat caudate putamen,and the average diffusion speeds of LY and Gd-DTPA were: (1.03±0.29)×10(-3) mm2/s, (0.81±0.27)×10(-3) mm2/s, respectively, no significant difference was demonstrated (t=0.759, P=0.490); half-time of single intensity of LY and Gd-DTPA was (2.58±0.04) h, (2.46±0.10) h, respectively, no significant difference was found (t=2.025, P=0.113). The diffusion area ratios between LY and Gd-DTPA in rat caudate putamen was not statistically different at hours 0.5, 1, 2, 3 and 7 (t=2.249, P=0.088; t=2.582, P=0.061; t=1.966, P=0.121; t=0.132, P=0.674; t=0.032, P=0.976), while, a slightly difference was found at 11 h (t=2.917, P=0.043,in analysis by t test). LY present the same diffusion property with Gd-DTPA in porous media witch including agarose gels and live rat brain tissue, indicates that LY is a suitable fluorescent probe for optical imaging of brain ISS, and it can be used for microscopic, macro and in vitro measure of brain ISS.

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis.

PubMed

Louws, F J; Bell, J; Medina-Mora, C M; Smart, C D; Opgenorth, D; Ishimaru, C A; Hausbeck, M K; de Bruijn, F J; Fulbright, D W

1998-08-01

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.

Influence of amino acids Shiff bases on irradiated DNA stability in vivo.

PubMed

Karapetyan, N H; Malakyan, M H; Bajinyan, S A; Torosyan, A L; Grigoryan, I E; Haroutiunian, S G

2013-01-01

To reveal protective role of the new Mn(II) complexes with Nicotinyl-L-Tyrosinate and Nicotinyl-L-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-L-Tyrosinate) and Mn(II)(Nicotinil-L-Tryptophanate)2 improves the DNA characteristics.

Agarose-based biomaterials for tissue engineering.

PubMed

Zarrintaj, Payam; Manouchehri, Saeed; Ahmadi, Zahed; Saeb, Mohammad Reza; Urbanska, Aleksandra M; Kaplan, David L; Mozafari, Masoud

2018-05-01

Agarose is a natural polysaccharide polymer having unique characteristics that give reason to consider it for tissue engineering applications. Special characteristics of agarose such as its excellent biocompatibility, thermo-reversible gelation behavior and physiochemical features support its use as a biomaterial for cell growth and/or controlled/localized drug delivery. The resemblance of this natural carbohydrate polymer to the extracellular matrix results in attractive features that bring about a strong interest in its usage in the field. The scope of this review is to summarize the extensive researches addressing agarose-based biomaterials in order to provide an in-depth understanding of its tissue engineering-related applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

An automated microplate-based method for monitoring DNA strand breaks in plasmids and bacterial artificial chromosomes

PubMed Central

Rock, Cassandra; Shamlou, Parviz Ayazi; Levy, M. Susana

2003-01-01

A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r2 > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of <5% coefficient of variation. Automation of the method showed equivalence to the manual procedure with high reproducibility and low variability within wells. The method described requires as little as 0.5 ng of DNA per well and a 96-well microplate can be analysed in 12 min providing an attractive option for analysis of high molecular weight vectors. A preparation of a 116 kb bacterial artificial chromosome was subjected to chemical and shear degradation and DNA integrity was tested using the method. Good correlation was obtained between time of chemical degradation and shear rate with fluorescence response. Results obtained from pulsed- field electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method. PMID:12771229

Centered reduced moments and associate density functions applied to alkaline comet assay.

PubMed

Castaneda, Roman; Pelaez, Alejandro; Marquez, Maria-Elena; Abad, Pablo

2005-01-01

The single cell gel electrophoresis assay is a sensitive, rapid, and visual technique for deoxyribonucleic acid (DNA) strand-break detection in individual mammalian cells, whose application has significantly increased in the past few years. The cells are embedded in agarose on glass slides followed by lyses of the cell membrane. Thereafter, damaged DNA strands are electrophoresed away from the nucleus towards the anode giving the appearance of a comet tail. Nowadays, charge coupled device cameras are attached at optical microscopes for recording the images of the cells, and digital image processing is applied for obtaining quantitative descriptors. However, the conventional software is usually expensive, inflexible and, in many cases, can only provide low-order descriptors based in image segmentation, determination of centers of mass, and Euclidean distances. Associated density functions and centered reduced moments offer an effective and flexible alternative for quantitative analysis of the comet cells. We will show how the position of the center of mass, the lengths and orientation of the main semiaxes, and the eccentricity of such images can be accurately determined by this method.

The effect of solute concentration on hindered gradient diffusion in polymeric gels

NASA Astrophysics Data System (ADS)

Buck, Kristan K. S.; Dungan, Stephanie R.; Phillips, Ronald J.

1999-10-01

The effect of solute concentration on hindered diffusion of sphere-like colloidal solutes in stiff polymer hydrogels is examined theoretically and experimentally. In the theoretical development, it is shown that the presence of the gel fibres enhances the effect of concentration on the thermodynamic driving force for gradient diffusion, while simultaneously reducing the effect of concentration on the hydrodynamic drag. The result is that gradient diffusion depends more strongly on solute concentration in gels than it does in pure solution, by an amount that depends on the partition coefficient and hydraulic permeability of the gel solute system. Quantitative calculations are made to determine the concentration-dependent diffusivity correct to first order in solute concentration. In order to compare the theoretical predictions with experimental data, rates of diffusion have been measured for nonionic micelles and globular proteins in solution and agarose hydrogels at two gel concentrations. The measurements were performed by using holographic interferometry, through which one monitors changes in refractive index as gradient diffusion takes place within a transparent gel. If the solutes are modelled as spheres with short-range repulsive interactions, then the experimentally measured concentration dependence of the diffusivities of both the protein and micelles is in good agreement with the theoretical predictions.

Towards the feasibility of using ultrasound to determine mechanical properties of tissues in a bioreactor

PubMed Central

Mansour, Joseph M.; Gu, Di-Win Marine; Chung, Chen-Yuan; Heebner, Joseph; Althans, Jake; Abdalian, Sarah; Schluchter, Mark D.; Liu, Yiying; Welter, Jean F.

2016-01-01

Introduction Our ultimate goal is to non-destructively evaluate mechanical properties of tissue-engineered (TE) cartilage using ultrasound (US). We used agarose gels as surrogates for TE cartilage. Previously, we showed that mechanical properties measured using conventional methods were related to those measured using US, which suggested a way to non-destructively predict mechanical properties of samples with known volume fractions. In this study, we sought to determine whether the mechanical properties of samples, with unknown volume fractions could be predicted by US. Methods Aggregate moduli were calculated for hydrogels as a function of SOS, based on concentration and density using a poroelastic model. The data were used to train a statistical model, which we then used to predict volume fractions and mechanical properties of unknown samples. Young's and storage moduli were measured mechanically. Results The statistical model generally predicted the Young's moduli in compression to within < 10% of their mechanically measured value. We defined positive linear correlations between the aggregate modulus predicted from US and both the storage and Young's moduli determined from mechanical tests. Conclusions Mechanical properties of hydrogels with unknown volume fractions can be predicted successfully from US measurements. This method has the potential to predict mechanical properties of TE cartilage non-destructively in a bioreactor. PMID:25092421

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations

NASA Astrophysics Data System (ADS)

Hosny, Neveen A.; Lee, David A.; Knight, Martin M.

2012-01-01

Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)3]2+ characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Single photon counting fluorescence lifetime detection of pericellular oxygen concentrations.

PubMed

Hosny, Neveen A; Lee, David A; Knight, Martin M

2012-01-01

Fluorescence lifetime imaging microscopy offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods are either invasive, require custom-made systems, or show limited spatial resolution. Therefore, these methods are unsuitable for investigation of pericellular oxygen concentrations. This study describes an adaptation of commercially available equipment which has been optimized for quantitative extracellular oxygen detection with high lifetime accuracy and spatial resolution while avoiding systematic photon pile-up. The oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)(3)](2+), was excited using a two-photon excitation laser. Lifetime was measured using a Becker & Hickl time-correlated single photon counting, which will be referred to as a TCSPC card. [Ru(bipy)(3)](2+) characterization studies quantified the influences of temperature, pH, cellular culture media and oxygen on the fluorescence lifetime measurements. This provided a precisely calibrated and accurate system for quantification of pericellular oxygen concentration based on measured lifetimes. Using this technique, quantification of oxygen concentrations around isolated viable chondrocytes, seeded in three-dimensional agarose gel, revealed a subpopulation of cells that exhibited significant spatial oxygen gradients such that oxygen concentration reduced with increasing proximity to the cell. This technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

A simple method for MR elastography: a gradient-echo type multi-echo sequence.

PubMed

Numano, Tomokazu; Mizuhara, Kazuyuki; Hata, Junichi; Washio, Toshikatsu; Homma, Kazuhiro

2015-01-01

To demonstrate the feasibility of a novel MR elastography (MRE) technique based on a conventional gradient-echo type multi-echo MR sequence which does not need additional bipolar magnetic field gradients (motion encoding gradient: MEG), yet is sensitive to vibration. In a gradient-echo type multi-echo MR sequence, several images are produced from each echo of the train with different echo times (TEs). If these echoes are synchronized with the vibration, each readout's gradient lobes achieve a MEG-like effect, and the later generated echo causes a greater MEG-like effect. The sequence was tested for the tissue-mimicking agarose gel phantoms and the psoas major muscles of healthy volunteers. It was confirmed that the readout gradient lobes caused an MEG-like effect and the later TE images had higher sensitivity to vibrations. The magnitude image of later generated echo suffered the T2 decay and the susceptibility artifacts, but the wave image and elastogram of later generated echo were unaffected by these effects. In in vivo experiments, this method was able to measure the mean shear modulus of the psoas major muscle. From the results of phantom experiments and volunteer studies, it was shown that this method has clinical application potential. Copyright © 2014 Elsevier Inc. All rights reserved.

Genomic Inprinting of the M6P/IGF2 Receptor: A Novel Breast Cancer Susceptibility Mechanism

DTIC Science & Technology

2001-07-01

and 72°Cx60 s for 35 cycles). Amplification products were purified in heterozygous 3-day-old embryos (n=17, data not from agarose gels using ...colugo), malian phylogeny, these sequences were used in addition to pre- AF339885 ( pig ), AF342813 (opossum), and AF342814 (echidna). viously...were varied and 973 were Ind.). Typical PCR reactions used 1% of the RNA-to-cDNA RT products , parsimony informative. We report herein only the analyses

Plasmid Isolation in Legionella pneumophila and Legionella-like Organisms.

DTIC Science & Technology

1980-08-22

hag, to our knowledge, not been reported. However in view of the narrow spectrum of antibiotics effective in the treatment of diseases caused by L...8217 disease bacterium isolated in 1947. Ann. Intern. Med. 90:659-661. 16. Meyers, J. A., D. Sanchez, L. P. Elwell, and S . Falkow. 1976. Simple agarose gel...AD-A090 844 ARMY MEDICAL RESEARCH INST OF INFECTIOUS DISEASES FR-ETC FIG 6/5 PLASMID ISOLATI ON IN LEGIONELLA PNEUMOPHILA AND LEGIONELLA-LIKE--ETC(U

Characterization of the Origin of DNA Replication of the Coxiella burnetii Chromosome

DTIC Science & Technology

1990-01-26

chromosomal DNAs (FIG. IB): the 19.4-kb EcoR I fragment of Salmonella typhimurium DNA (lane 4),9 the 17.5-kb Sal I fragment of Enterobacter aerogenes ...IacZYA-argF) U 1694680d IacZAM15 Salmonella typhimurium Wild type WVUd Kiebsiella pneumoniae Wild type WVUd Enterobacter aero genes Wild type WVUd... aerogenes and K. pneumoniae were digested with appropriate restriction enzymes. The restriction fragments were separated on a 0.9% agarose gel, transferred to

Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to evaluate von Willebrand factor protease activity.

PubMed

Kirzek, D M; Rick, M E

2001-03-01

A method for evaluating the activity of the von Willebrand factor (vWF) protease is described, and a clinical application is illustrated. The procedure utilizes gel electrophoresis, Western blotting, and luminographic detection methods to evaluate the distribution of vWF multimers before and after incubation of clinical samples under conditions that favor proteolysis by this enzyme. Physiologically, the high-molecular-weight multimers of vWF are cleaved by the vWF protease under conditions of high shear stress in parts of the arterial circulation; cleavage of vWF multimers is also observed after exposure of vWF to denaturing agents in vitro and thus can serve as a laboratory test for the activity of the protease. vWF protease activity is decreased or absent in patients with thrombotic thrombocytopenic purpura due to an inhibiting autoantibody, and this leads to high levels of noncleaved vWF and to life-threatening thrombosis, thrombocytopenia and anemia. The assay evaluates the activity of the protease by assessing the cleavage of vWF multimers after patient plasmas are incubated in vitro under denaturing conditions. With the use of these electrophoresis and Western blotting techniques, patient plasmas can be rapidly assessed for the activity of the vWF protease which may aid in the treatment strategy for these patients.

A novel superporous agarose medium for high-speed protein chromatography.

PubMed

Shi, Qing-Hong; Zhou, Xin; Sun, Yan

2005-12-05

A novel superporous agarose (SA) bead characterized by the presence of wide pores has been fabricated by water-in-oil emulsification using solid granules of calcium carbonate as porogenic agent. After cross-linking, the solid granules were removed by dissolving them in hydrochloric acid. Then, the gel was modified with diethylaminoethyl groups to create an anion exchanger, SA-DEAE, for protein adsorption. A homogeneous agarose (HA) bead was also produced and modified with DEAE for comparison. It was found that the porosity of SA-DEAE was about 6% larger than that of HA-DEAE. Moreover, both optical micrographs and confocal laser scanning microscopy (CLSM) of the ion exchangers with adsorbed fluorescein isothiocyanate (FITC) labeled IgG revealed the superporous structure of the SA medium. In addition, the SA-DEAE column had lower backpressure than the HA-DEAE column, confirming the convective flow of mobile phase through the wide pores. Due to the presence of the wide pores, more channels were available for protein transport and, furthermore, more diffusive pores in the agarose network were accessible for the protein approach from different directions. This led to 40% higher protein capacity and two times higher effective pore diffusivity in the SA-DEAE than in HA-DEAE. Moreover, an increase of the efficiency of the SA-DEAE column until a flow rate of 5 cm/min and the independency of the column efficiency at flow rates from 5 to 17.8 cm/min was found, indicating that intraparticle mass transfer was intensified by convective flow at elevated flow rates. Therefore, the chromatographic resolution of IgG and BSA was little affected up to a flow rate of 17.8 cm/min. The results indicate that the SA medium is favorable for high-speed protein chromatography. (c) 2005 Wiley Periodicals, Inc.

Application of immuno-PCR assay for the detection of serum IgE specific to Bermuda allergen.

PubMed

Rahmatpour, Samine; Khan, Amjad Hayat; Nasiri Kalmarzi, Rasoul; Rajabibazl, Masoumeh; Tavoosidana, Gholamreza; Motevaseli, Elahe; Zarghami, Nosratollah; Sadroddiny, Esmaeil

2017-04-01

In vivo and in vitro tests are the two major ways of identifying the triggering allergens in sensitized individuals with allergic symptoms. Both methods are equally significant in terms of sensitivity and specificity. However, in certain circumstances, in vitro methods are highly preferred because they circumvent the use of sensitizing drugs in patients. In current study, we described a highly sensitive immuno-PCR (iPCR) assay for serum IgE specific to Bermuda allergens. Using oligonucleotide-labelled antibody, we used iPCR for the sensitive detection of serum IgE. The nucleotide sequence was amplified using conventional PCR and the bands were visualized on 2.5% agarose gel. Results demonstrated a 100-fold enhancement in sensitivity of iPCR over commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our iPCR method was highly sensitive for Bermuda-specific serum IgE and could be beneficial in allergy clinics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Assay of mucins in human tear fluid.

PubMed

Spurr-Michaud, Sandra; Argüeso, Pablo; Gipson, Ilene

2007-05-01

Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r2 > 0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention.

Assay of Mucins in Human Tear Fluid

PubMed Central

Spurr-Michaud, Sandra; Argüeso, Pablo; Gipson, Ilene

2007-01-01

Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r2 >0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention. PMID:17399701

A medical device-grade T1 and ECV phantom for global T1 mapping quality assurance-the T1 Mapping and ECV Standardization in cardiovascular magnetic resonance (T1MES) program.

PubMed

Captur, Gabriella; Gatehouse, Peter; Keenan, Kathryn E; Heslinga, Friso G; Bruehl, Ruediger; Prothmann, Marcel; Graves, Martin J; Eames, Richard J; Torlasco, Camilla; Benedetti, Giulia; Donovan, Jacqueline; Ittermann, Bernd; Boubertakh, Redha; Bathgate, Andrew; Royet, Celine; Pang, Wenjie; Nezafat, Reza; Salerno, Michael; Kellman, Peter; Moon, James C

2016-09-22

T 1 mapping and extracellular volume (ECV) have the potential to guide patient care and serve as surrogate end-points in clinical trials, but measurements differ between cardiovascular magnetic resonance (CMR) scanners and pulse sequences. To help deliver T 1 mapping to global clinical care, we developed a phantom-based quality assurance (QA) system for verification of measurement stability over time at individual sites, with further aims of generalization of results across sites, vendor systems, software versions and imaging sequences. We thus created T1MES: The T1 Mapping and ECV Standardization Program. A design collaboration consisting of a specialist MRI small-medium enterprise, clinicians, physicists and national metrology institutes was formed. A phantom was designed covering clinically relevant ranges of T 1 and T 2 in blood and myocardium, pre and post-contrast, for 1.5 T and 3 T. Reproducible mass manufacture was established. The device received regulatory clearance by the Food and Drug Administration (FDA) and Conformité Européene (CE) marking. The T1MES phantom is an agarose gel-based phantom using nickel chloride as the paramagnetic relaxation modifier. It was reproducibly specified and mass-produced with a rigorously repeatable process. Each phantom contains nine differently-doped agarose gel tubes embedded in a gel/beads matrix. Phantoms were free of air bubbles and susceptibility artifacts at both field strengths and T 1 maps were free from off-resonance artifacts. The incorporation of high-density polyethylene beads in the main gel fill was effective at flattening the B 1 field. T 1 and T 2 values measured in T1MES showed coefficients of variation of 1 % or less between repeat scans indicating good short-term reproducibility. Temperature dependency experiments confirmed that over the range 15-30 °C the short-T 1 tubes were more stable with temperature than the long-T 1 tubes. A batch of 69 phantoms was mass-produced with random sampling of ten of these showing coefficients of variations for T 1 of 0.64 ± 0.45 % and 0.49 ± 0.34 % at 1.5 T and 3 T respectively. The T1MES program has developed a T 1 mapping phantom to CE/FDA manufacturing standards. An initial 69 phantoms with a multi-vendor user manual are now being scanned fortnightly in centers worldwide. Future results will explore T 1 mapping sequences, platform performance, stability and the potential for standardization.

Genetic polymorphisms variants in interleukin-6 and interleukin-1beta patients with obstructive sleep apnea syndrome in East Northern Turkey.

PubMed

Gok, Ilhami; Huseyinoglu, Nergiz; Ilhan, Dogan

2015-08-01

To investigate the relationship of IL-1β and IL-6 cytokine gene polymorphisms with obstructive sleep apnea syndrome (OSAS) in 61 patients admitted to the neurology clinic in Kafkas University Hospital with insomnia problem who were diagnosed with OSAS in sleeping labs, and 80 healthy subjects not associated with the syndrome. METHODS :Blood samples were taken to isolate DNA from patients diagnosed with OSAS based on polysomnography results and healthy controls. DNA amplification of the genes was performed with PCR. Amplification products were cut with the restriction enzymes in order to determine IL-1 gene (TaqI) and IL-6 gene (Lwel) polymorphisms. The cut DNA fragments were carried out in agarose gel electrophoresis, and RFLP analysis was performed by utilizing the images with gel imaging system. PCR products were sequenced with an Applied Biosystems Automated Sequencer. Polymorphic changes were observed for IL-1β gene in 26 of 62 patients (41.9%), and 16 of the 80 (25.8%) in the control group. The incidence of polymorphic changes in IL-6 gene was in seen in seven (of the 62 patients) (11.3%), and in the 16 (20%) controls. The findings on the genomic level in OSAS may provide an important contribution to diagnosis of obstructive sleep apnea syndrome in clinical practice, as well as it helps to obtain the results easily about environmental and genetic interaction of OSAS patients. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

An anthropomorphic multimodality (CT/MRI) head phantom prototype for end-to-end tests in ion radiotherapy.

PubMed

Gallas, Raya R; Hünemohr, Nora; Runz, Armin; Niebuhr, Nina I; Jäkel, Oliver; Greilich, Steffen

2015-12-01

With the increasing complexity of external beam therapy "end-to-end" tests are intended to cover every step from therapy planning through to follow-up in order to fulfill the higher demands on quality assurance. As magnetic resonance imaging (MRI) has become an important part of the treatment process, established phantoms such as the Alderson head cannot fully be used for those tests and novel phantoms have to be developed. Here, we present a feasibility study of a customizable multimodality head phantom. It is initially intended for ion radiotherapy but may also be used in photon therapy. As basis for the anthropomorphic head shape we have used a set of patient computed tomography (CT) images. The phantom recipient consisting of epoxy resin was produced by using a 3D printer. It includes a nasal air cavity, a cranial bone surrogate (based on dipotassium phosphate), a brain surrogate (based on agarose gel), and a surrogate for cerebrospinal fluid (based on distilled water). Furthermore, a volume filled with normoxic dosimetric gel mimicked a tumor. The entire workflow of a proton therapy could be successfully applied to the phantom. CT measurements revealed CT numbers agreeing with reference values for all surrogates in the range from 2 HU to 978 HU (120 kV). MRI showed the desired contrasts between the different phantom materials especially in T2-weighted images (except for the bone surrogate). T2-weighted readout of the polymerization gel dosimeter allowed approximate range verification. Copyright © 2015. Published by Elsevier GmbH.

Identification of a haptoglobin-hemoglobin complex in the Alaskan Least Cisco (Coregonus sardinella).

PubMed

Wahl, S M; Boger, J K; Michael, V; Duffy, L K

1992-01-01

The hemoglobin and a hemoglobin binding protein have been characterized in the Arctic fish (Coregonus sardinella). The evolutionary significance of the hemoglobin and plasma protein differences between fish and mammals is still unresolved. Blood samples from the Alaskan Least Cisco were separated into plasma and hemoglobin fractions and the proteins in these fractions were analyzed both by alkaline agarose gel electrophoresis, by isolelectric focusing, and by capillary electrophoresis. Staining the plasma proteins gels with o-dianisidine revealed hemoglobin containing protein complexes. A hemoglobin-containing band was observed in hemolyzed plasma which did not migrate with free hemoglobin, and is believed to be hemoglobin-haptoglobin complex. Size exclusion chromatography further characterized the hemoglobin as disassociating freely into dimers, and hemoglobin-haptoglobin complex having a molecular weight greater then 200,000 daltons.

Simple and Inexpensive but Highly Discriminating Method for Computer-Assisted DNA Fingerprinting of Pseudomonas aeruginosa

PubMed Central

Al-Samarrai, Taha H.; Zhang, Ningxin; Lamont, Iain L.; Martin, Lois; Kolbe, John; Wilsher, Margaret; Morris, Arthur J.; Schmid, Jan

2000-01-01

We describe here a method for computer-assisted fingerprinting of Pseudomonas aeruginosa. In this method, DNA is digested with SalI, and bands with molecular sizes of ≥9.7 kb are visually scored after electrophoresis on agarose gels. Pattern scores are entered into a Microsoft Excel database. In scoring, the number of bands within each of a set of molecular size ranges is scored, rather than the absolute molecular size of each band, substantially enhancing the speed and reproducibility of the method, while eliminating the need for using expensive gel scanning equipment and software. Pattern scores are used to generate matrices of genetic distance values, which can be visualized in neighbor-joining trees. The method reliably distinguishes two epidemiologically unrelated isolates in 99.3% of all comparisons. The genetic relationships between isolates observed with the method were consistent with those obtained by analysis of two P. aeruginosa genes, indicating that it provides valid estimates of genetic divergence between isolates. Using the method, respiratory tract isolates from cystic fibrosis patients in Green Lane Hospital in Auckland, New Zealand, were shown to be genetically less diverse than epidemiologically unrelated isolates from other patients. This finding was not due to the existence of clusters of related strains specialized toward colonization of the respiratory tract and thus was indicative of transmission between patients. Analysis of multiple isolates from individual cystic fibrosis patients suggested that up to five separate clusters of genetically related strains may simultaneously be present in a patient. The method described should significantly enhance our ability to investigate the epidemiology of P. aeruginosa. PMID:11101578

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

PubMed

Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution analysis in inhibitory tissues.

Wavefront sensorless adaptive optics temporal focusing-based multiphoton microscopy

PubMed Central

Chang, Chia-Yuan; Cheng, Li-Chung; Su, Hung-Wei; Hu, Yvonne Yuling; Cho, Keng-Chi; Yen, Wei-Chung; Xu, Chris; Dong, Chen Yuan; Chen, Shean-Jen

2014-01-01

Temporal profile distortions reduce excitation efficiency and image quality in temporal focusing-based multiphoton microscopy. In order to compensate the distortions, a wavefront sensorless adaptive optics system (AOS) was integrated into the microscope. The feedback control signal of the AOS was acquired from local image intensity maximization via a hill-climbing algorithm. The control signal was then utilized to drive a deformable mirror in such a way as to eliminate the distortions. With the AOS correction, not only is the axial excitation symmetrically refocused, but the axial resolution with full two-photon excited fluorescence (TPEF) intensity is also maintained. Hence, the contrast of the TPEF image of a R6G-doped PMMA thin film is enhanced along with a 3.7-fold increase in intensity. Furthermore, the TPEF image quality of 1μm fluorescent beads sealed in agarose gel at different depths is improved. PMID:24940539

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

PubMed

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.

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

PubMed Central

Pourjahed, Atefeh; Rabiee, Mohammad; Tahriri, Mohammadreza

2013-01-01

Objective(s): Microarrays are potential analyzing tools for genomics and proteomics researches, which is in needed of suitable substrate for coating and also hybridization of biomolecules. Materials and Methods: In this research, a thin film of oxidized agarose was prepared on the glass slides which previously coated with poly-L-lysine (PLL). Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups; also bound to the amino groups of biomolecules. These linkages were fixed by UV irradiation. The prepared substrates were compared to only agarose-coated and PLL-coated slides. Results: Results on atomic force microscope (AFM) demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. In addition, the signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively. Conclusion: 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. PMID:24570832

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

PubMed

Cusick, M E

1992-12-29

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

Electrophoretic properties of BSA-coated quantum dots.

PubMed

Bücking, Wendelin; Massadeh, Salam; Merkulov, Alexei; Xu, Shu; Nann, Thomas

2010-02-01

Low toxic InP/ZnS quantum dots (QDs), ZnS:Mn(2+)/ZnS nanocrystals and CdSe/ZnS nanoparticles were rendered water-dispersible by different ligand-exchange methods. Eventually, they were coated with bovine serum albumin (BSA) as a model protein. All particles were characterised by isotachophoresis (ITP), laser Doppler velocimetry (LDV) and agarose gel electrophoresis. It was found that the electrophoretic mobility and colloidal stability of ZnS:Mn(2+)/ZnS and CdSe/ZnS nanoparticles, which bore short-chain surface ligands, was primarily governed by charges on the nanoparticles, whereas InP/ZnS nanocrystals were not charged per se. BSA-coated nanoparticles showed lower electrophoretic mobility, which was attributed to their larger size and smaller overall charge. However, these particles were colloidally stable. This stability was probably caused by steric stabilisation of the BSA coating.

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

PubMed

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 s

Inactivation of Vibrio parahaemolyticus by antimicrobial photodynamic technology using methylene blue.

PubMed

Deng, Xi; Tang, Shuze; Wu, Qian; Tian, Juan; Riley, William W; Chen, Zhenqiang

2016-03-30

Vibrio parahaemolyticus is the leading causative pathogen of gastroenteritis often related to contaminated seafood. Photodynamic inactivation has been recently proposed as a strategy for killing cells and viruses. The objective of this study was to verify the bactericidal effects caused by photodynamic inactivation using methylene blue (MB) over V. parahaemolyticus via flow cytometry, agarose gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Vibrio parahaemolyticus counts were determined using the most probable number method. A scanning electron microscope and a transmission electron microscope were employed to intuitively analyze internal and external cell structure. Combination of MB and laser treatment significantly inhibited the growth of V. parahaemolyticus. The inactivation rate of V. parahaemolyticus was >99.99% and its counts were reduced by 5 log10 in the presence of 0.05 mg mL(-1) MB when illuminated with visible light (power density 200 mW cm(-2)) for 25 min. All inactivated cells showed morphological changes, leakage of cytoplasm and degradation of protein and DNA. Results from this study indicated that photodynamic technology using MB produced significant inactivation of V. parahaemolyticus mainly brought about by the degradation of protein and DNA. © 2015 Society of Chemical Industry.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

Investigations of nanoscale variations in spin and charge transport in manganites and organic semiconductors using spin polarized scanning tunneling spectroscopy

NASA Astrophysics Data System (ADS)

Hughes, Cameron Richard

Analysis of DNA structure and behavior, up to and including full sequencing of a genome's bases, and of biological processes such as replication, transcription and translation, is essential for an understanding of genetic variation, heritable diseases and the effects of environmental factors. Recently, single-molecule techniques have been developed to study DNA properties in unprecedented detail. For a number of these techniques, controlled adsorption of linearly stretched DNA molecules on surfaces is necessary. In experiments where hybridization of adsorbed molecules to labeled probes is used to determine DNA structure, single-stranded DNA is needed. Conventionally, for long DNA's (up to Mbp), double-stranded DNA is deposited on a surface and denatured in-situ. While successful, this method has several disadvantages. This thesis reports efforts to directly adsorb long single-stranded DNA's out of solution as an alternative strategy. It consists of three parts: (1) Establishment of a simple method using Acridine Orange (AO) staining dye to determine whether DNA's are ss or ds on the surface. The method allows for the assessment of the degree of renaturation during deposition. Incubation of surface-adsorbed DNA in solutions of AO dye in the concentration range of 10--15uM were found to be effective for discriminating between ss DNA and ds DNA based on differences in the fluorescence emission spectra. (2) Deposition of ss DNA produced by heat denaturation on polymer-coated surfaces. Lambda DNA (48502bp) was adsorbed by drop evaporation or dipping/extraction of surface out of a buffered solution. The efficiency of deposition was optimized with respect to DNA concentration, buffer type and pH. (3) Separation of complementary single strands of Lambda, mono-cut digest and HindIII digest by gel electrophoresis. Using agarose gels in concentrations ranging from 0.4% to 1.4% (weight/volume), electric fields in the range 1--4V/cm in 1x Tris-Acetate-EDTA (TAE) buffer, good strand separation could be obtained. Both DC and pulsed electric fields were used and compared. Following separation, sense and anti-sense strands of lambda DNA were extracted from gels and deposited separately onto surfaces, and length distributions of the isolated molecules were measured by fluorescence microscopy.

Application of PCR to a clinical and environmental investigation of a case of equine botulism.

PubMed

Szabo, E A; Pemberton, J M; Gibson, A M; Thomas, R J; Pascoe, R R; Desmarchelier, P M

1994-08-01

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples.

Application of PCR to a clinical and environmental investigation of a case of equine botulism.

PubMed Central

Szabo, E A; Pemberton, J M; Gibson, A M; Thomas, R J; Pascoe, R R; Desmarchelier, P M

1994-01-01

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples. Images PMID:7989554

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick.

PubMed

Wang, H; Sun, M; Xu, D; Podok, P; Xie, J; Jiang, Ys; Lu, Lq

2018-05-28

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV-2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV-2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV-2. The highly conserved ORF72 of CyHV-2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA-LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross-reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA-LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV-2 when resources are limited. © 2018 John Wiley & Sons Ltd.

A model for the salt effect on adsorption equilibrium of basic protein to dye-ligand affinity adsorbent.

PubMed

Zhang, Songping; Sun, Yan

2004-01-01

A model describing the salt effect on adsorption equilibrium of a basic protein, lysozyme, to Cibacron Blue 3GA-modified Sepharose CL-6B (CB-Sepharose) has been developed. In this model, it is assumed that the presence of salt causes a fraction of dye-ligand molecules to lodge to the surface of the agarose gel, resulting from the induced strong hydrophobic interaction between dye ligand and agarose matrix. The salt effect on the lodging of dye-ligand is expressed by the equilibrium between salt and dye-ligand. For the interactions between protein and vacant binding sites, stoichiometric equations based either on cation exchanges or on hydrophobic interactions are proposed since the CB dye can be regarded as a cation exchanger contributed by the sulfonate groups on it. Combining with the basic concept of steric mass-action theory for ion exchange, which considers both the multipoint nature and the macromolecular steric shielding of protein adsorption, an explicit isotherm for protein adsorption equilibrium on the dye-ligand adsorbent is formulated, involving salt concentration as a variable. Analysis of the model parameters has yielded better understanding of the mechanism of salt effects on adsorption of the basic protein. Moreover, the model predictions are in good agreement with the experimental data over a wide range of salt and ligand concentrations, indicating the predictive nature of the model.

Clusters of DNA damage induced by ionizing radiation: formation of short DNA fragments. II. Experimental detection

NASA Technical Reports Server (NTRS)

Rydberg, B.; Chatterjee, A. (Principal Investigator)

1996-01-01

The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.

Novel hydrophobic interaction chromatography matrix for specific isolation and simple elution of immunoglobulins (A, G, and M) from porcine serum.

PubMed

Ramos-Clamont, Gabriela; del Carmen Candia-Plata, Maria; Zamudio, Roberto Guzman; Vazquez-Moreno, Luz

2006-07-28

A new, highly acetylated agarose matrix (HA-Sepharose) was synthesized and used as a hydrophobic interaction chromatography (HIC) medium to specifically isolate immunoglobulins (Igs) from porcine serum. Recovery of Igs was in a single step and under mild conditions. HA-Sepharose adsorption was studied in terms of salt, gel acetylation time, flow rate, and protein concentration on the loading buffer. At 0.5 M Na2SO4, control with unmodified Sepharose retained a small fraction (0.70 mg/mL of matrix) of serum albumin. On the contrary HA-Sepharose retained primary Igs (IgA, IgG, and 53% of IgM) as revealed by sodium dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS-PAGE), quantitative radial immunodiffusion and immunodetection. At a flow rate of 1 mL/min, the HA-Sepharose column capacity (3.9 mg/mL of matrix) was similar to the reported capacity for the commercial thiophilic T-gel. However, HA-Sepharose showed higher recovery of IgA and IgM than the T-gel in the same salt conditions, clearly an advantage in terms of immunoglobulin recovery strategies. Acetylation changed the matrix adsorption from albumin to immunoglobulins; thus, the highly acetylated gel rendered recoveries of Igs from unprocessed porcine serum practically free of albumin.

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

PubMed

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples

PubMed Central

Kimura, Richard; Mandrell, Robert E.; Galland, John C.; Hyatt, Doreene; Riley, Lee W.

2000-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in industrialized countries. We developed a rapid and simple test for detecting E. coli O157:H7 using a method based on restriction site polymorphisms. Restriction-site-specific PCR (RSS-PCR) involves the amplification of DNA fragments using primers based on specific restriction enzyme recognition sequences, without the use of endonucleases, to generate a set of amplicons that yield “fingerprint” patterns when resolved electrophoretically on an agarose gel. The method was evaluated in a blinded study of E. coli isolates obtained from environmental samples collected at beef cattle feedyards. The 54 isolates were all initially identified by a commonly used polyclonal antibody test as belonging to O157:H7 serotype. They were retested by anti-O157 and anti-H7 monoclonal antibody enzyme-linked immunosorbent assay (ELISA). The RSS-PCR method identified all 28 isolates that were shown to be E. coli O157:H7 by the monoclonal antibody ELISA as belonging to the O157:H7 serotype. Of the remaining 26 ELISA-confirmed non-O157:H7 strains, the method classified 25 strains as non-O157:H7. The specificity of the RSS-PCR results correlated better with the monoclonal antibody ELISA than with the polyclonal antibody latex agglutination tests. The RSS-PCR method may be a useful test to distinguish E. coli O157:H7 from a large number of E. coli isolates from environmental samples. PMID:10831431

Non-toxic agarose/gelatin-based microencapsulation system containing gallic acid for antifungal application.

PubMed

Lam, P-L; Gambari, R; Kok, S H-L; Lam, K-H; Tang, J C-O; Bian, Z-X; Lee, K K-H; Chui, C-H

2015-02-01

Aspergillus niger (A. niger) is a common species of Aspergillus molds. Cutaneous aspergillosis usually occurs in skin sites near intravenous injection and approximately 6% of cutaneous aspergillosis cases which do not involve burn or HIV-infected patients are caused by A. niger. Biomaterials and biopharmaceuticals produced from microparticle-based drug delivery systems have received much attention as microencapsulated drugs offer an improvement in therapeutic efficacy due to better human absorption. The frequently used crosslinker, glutaraldehyde, in gelatin-based microencapsulation systems is considered harmful to human beings. In order to tackle the potential risks, agarose has become an alternative polymer to be used with gelatin as wall matrix materials of microcapsules. In the present study, we report the eco-friendly use of an agarose/gelatin-based microencapsulation system to enhance the antifungal activity of gallic acid and reduce its potential cytotoxic effects towards human skin keratinocytes. We used optimal parameter combinations, such as an agarose/gelatin ratio of 1:1, a polymer/oil ratio of 1:60, a surfactant volume of 1% w/w and a stirring speed of 900 rpm. The minimum inhibitory concentration of microencapsulated gallic acid (62.5 µg/ml) was significantly improved when compared with that of the original drug (>750 µg/ml). The anti-A. niger activity of gallic acid -containing microcapsules was much stronger than that of the original drug. Following 48 h of treatment, skin cell survival was approximately 90% with agarose/gelatin microcapsules containing gallic acid, whereas cell viability was only 25-35% with free gallic acid. Our results demonstrate that agarose/gelatin-based microcapsules containing gallic acid may prove to be helpful in the treatment of A. niger-induced skin infections near intravenous injection sites.

Physical characterization of neurocatheter performance in a brain phantom gelatin with nanoscale porosity: steady-state and oscillatory flows

NASA Astrophysics Data System (ADS)

Bauman, M. A.; Gillies, G. T.; Raghavan, R.; Brady, M. L.; Pedain, C.

2004-01-01

An agarose gelatin having nanoscale transport properties similar to those of in vivo mammalian brain was employed as a surrogate for living brain tissue in the evaluation of infusion therapy protocols and neurocatheters to be used in the treatment of brain tumours. The catheters under study were a polyimide tube of 950 µm outer diameter (OD) and 750 µm inner diameter (ID), and a silicone tube of 2.5 mm OD and 1.25 mm ID. From the pressure profiles that were measured during infusions of a solution of Bromphenol Blue dye into this gel, we infer that forces on the order of 0.1 fN were driving the solute molecules through the {\\approx } 200 nm intramatrix voids in the gel at rates of {\\approx } 10\

Towards the feasibility of using ultrasound to determine mechanical properties of tissues in a bioreactor.

PubMed

Mansour, Joseph M; Gu, Di-Win Marine; Chung, Chen-Yuan; Heebner, Joseph; Althans, Jake; Abdalian, Sarah; Schluchter, Mark D; Liu, Yiying; Welter, Jean F

2014-10-01

Our ultimate goal is to non-destructively evaluate mechanical properties of tissue-engineered (TE) cartilage using ultrasound (US). We used agarose gels as surrogates for TE cartilage. Previously, we showed that mechanical properties measured using conventional methods were related to those measured using US, which suggested a way to non-destructively predict mechanical properties of samples with known volume fractions. In this study, we sought to determine whether the mechanical properties of samples, with unknown volume fractions could be predicted by US. Aggregate moduli were calculated for hydrogels as a function of SOS, based on concentration and density using a poroelastic model. The data were used to train a statistical model, which we then used to predict volume fractions and mechanical properties of unknown samples. Young's and storage moduli were measured mechanically. The statistical model generally predicted the Young's moduli in compression to within <10% of their mechanically measured value. We defined positive linear correlations between the aggregate modulus predicted from US and both the storage and Young's moduli determined from mechanical tests. Mechanical properties of hydrogels with unknown volume fractions can be predicted successfully from US measurements. This method has the potential to predict mechanical properties of TE cartilage non-destructively in a bioreactor.

Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

NASA Astrophysics Data System (ADS)

Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

2018-04-01

ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

Partial Purification and Characterization of Restriction Endonuclease from Neisseria meningitidis.

DTIC Science & Technology

1983-12-01

by centrifugation at 15,000 x g for 10 min. Preparation of Cell-Free Extract The cell pellet was suspended in 10 mL of Tris-HCI buffer pH 7.6 (Tris, 20...free extract (CFE) was obtained by centrifugation at 100,000 x g for I h. To the CFE, glycerol was added to a final concentration of 10% and stored at... extract obtained from N. meningilidis when incubated with A DNA and analyzed by agarose gel electrophoresis did not give a clean fragmentation pattern

Genetic and Physiological Studies of Bacillus anthracis Related to Development of An Improved Vaccine

DTIC Science & Technology

1989-07-01

enzyme in a 1.5 ml Eppendorf tube. Appropriate amounts of distilled water and 1OX buffer were added to give a total volume of 100 pl. Reaction...mixtures were incubated in a 370 C water bath for 2 to 15 h. Digests were heated at 650C for 10 minutes to stop reactions and then resolved on agarose gels...430 C water bath with shaking (160 rpm) for 4 hours. The culture was transferred in a similar manner three additional times. Following the fourth

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

PubMed

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.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

PubMed

Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Introduction of structural affinity handles as a tool in selective nucleic acid separations

NASA Technical Reports Server (NTRS)

Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

2011-01-01

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card

NASA Astrophysics Data System (ADS)

Hosny, Neveen A.; Lee, David A.; Knight, Martin M.

2010-02-01

Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up. Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]2+, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.

Abstracts of papers presented at the 8th workshop of the Virology Section of the Deutsche Gesellschaft für Hygiene und Mikro-biologie, Würzburg, March 17-19, 1983.

PubMed

1983-09-01

17 adenovirus strains were found to be antigenically related to prototype Ad 15 by neutralization. No relationship to Ad 15, but to Ad 9 could be detected by hemagglutination-inhibition; we therefore named them Ad 15/H9 intermediate strains. After analysis of the genome by five different restriction enzymes, the fragment patterns obtained deviated widely from the prototype Ad 15, but only slightly from Ad 9. Differences could also be observed among the variants. After digestion by five restriction enzymes, altogether six genome types could be established among the 17 intermediate strains. To map the variations on the genome of the 15/H9 strains, two methods were employed: the double digestion of the DNA and DNA fragments together with the determination of the terminal fragments made it possible to construct a physical map. The second method depends on a particularity of adenoviruses: the DNA is covalently linked with a 55 kD protein at the 5' terminus. After digestion of the DNA, which does contain this protein, the terminal DNA fragments do not migrate into the agarose gel; after an additional digestion with pronase B, they do migrate into the gel. Thus the terminal fragments were determined by comparing the fragment patterns with and without previous pronase B treatment.

An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene

PubMed Central

Saluto, Alessandro; Brussino, Alessandro; Tassone, Flora; Arduino, Carlo; Cagnoli, Claudia; Pappi, Patrizia; Hagerman, Paul; Migone, Nicola; Brusco, Alfredo

2005-01-01

Several diagnostic strategies have been applied to the detection of FMR1 gene repeat expansions in fragile X syndrome. Here, we report a novel polymerase chain reaction-based strategy using the Expand Long Template PCR System (Roche Diagnostics, Mannheim, Germany) and the osmolyte betaine. Repeat expansions up to ∼330 CGGs in males and up to at least ∼160 CGGs in carrier women could be easily visualized on ethidium bromide agarose gels. We also demonstrated that fluorescence analysis of polymerase chain reaction products was a reliable tool to verify the presence of premutation and full mutation alleles both in males and in females. This technique, primarily designed to detect premutation alleles, can be used as a routine first screen for expanded FMR1 alleles. PMID:16258159

Thermal heterogeneity within aqueous materials quantified by 1H NMR spectroscopy: Multiparametric validation in silico and in vitro

NASA Astrophysics Data System (ADS)

Lutz, Norbert W.; Bernard, Monique

2018-02-01

We recently suggested a new paradigm for statistical analysis of thermal heterogeneity in (semi-)aqueous materials by 1H NMR spectroscopy, using water as a temperature probe. Here, we present a comprehensive in silico and in vitro validation that demonstrates the ability of this new technique to provide accurate quantitative parameters characterizing the statistical distribution of temperature values in a volume of (semi-)aqueous matter. First, line shape parameters of numerically simulated water 1H NMR spectra are systematically varied to study a range of mathematically well-defined temperature distributions. Then, corresponding models based on measured 1H NMR spectra of agarose gel are analyzed. In addition, dedicated samples based on hydrogels or biological tissue are designed to produce temperature gradients changing over time, and dynamic NMR spectroscopy is employed to analyze the resulting temperature profiles at sub-second temporal resolution. Accuracy and consistency of the previously introduced statistical descriptors of temperature heterogeneity are determined: weighted median and mean temperature, standard deviation, temperature range, temperature mode(s), kurtosis, skewness, entropy, and relative areas under temperature curves. Potential and limitations of this method for quantitative analysis of thermal heterogeneity in (semi-)aqueous materials are discussed in view of prospective applications in materials science as well as biology and medicine.

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

PubMed

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.

The first neural probe integrated with light source (blue laser diode) for optical stimulation and electrical recording.

PubMed

Park, HyungDal; Shin, Hyun-Joon; Cho, Il-Joo; Yoon, Eui-sung; Suh, Jun-Kyo Francis; Im, Maesoon; Yoon, Euisik; Kim, Yong-Jun; Kim, Jinseok

2011-01-01

In this paper, we report a neural probe which can selectively stimulate target neurons optically through Si wet etched mirror surface and record extracellular neural signals in iridium oxide tetrodes. Consequently, the proposed approach provides to improve directional problem and achieve at least 150/m gap distance between stimulation and recording sites by wet etched mirror surface in V-groove. Also, we developed light source, blue laser diode (OSRAM Blue Laser Diode_PL 450), integration through simple jig for one-touch butt-coupling. Furthermore, optical power and impedance of iridium oxide tetrodes were measured as 200 μW on 5 mW from LD and 206.5 k Ω at 1 kHz and we demonstrated insertion test of probe in 0.5% agarose-gel successfully. We have successfully transmitted a light of 450 nm to optical fiber through the integrated LD using by butt-coupling method.

A new microcolumn-type microchip for examining the expression of chimeric fusion genes using a nucleic acid sandwich hybridization technique.

PubMed

Ohnishi, Michihiro; Sasaki, Naoyuki; Kishimoto, Takuya; Watanabe, Hidetoshi; Takagi, Masatoshi; Mizutani, Shuki; Kishii, Noriyuki; Yasuda, Akio

2014-11-01

We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification. Copyright © 2014 Elsevier B.V. All rights reserved.

Mapping replication origins in yeast chromosomes.

PubMed

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.

The anti-human immunodeficiency virus agent 3'-fluorothymidine induces DNA damage and apoptosis in human lymphoblastoid cells.

PubMed Central

Sundseth, R; Joyner, S S; Moore, J T; Dornsife, R E; Dev, I K

1996-01-01

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo. PMID:8834875

Lens capsule structure assessed with atomic force microscopy

PubMed Central

Sueiras, Vivian M.; Moy, Vincent T.

2015-01-01

Purpose To image the ultrastructure of the anterior lens capsule at the nanoscale level using atomic force microscopy (AFM). Methods Experiments were performed on anterior lens capsules maintained in their in situ location surrounding the lens from six human cadavers (donor age range: 44–88 years), four cynomolgus monkeys (Macaca fascicularis age range: 4.83–8.92 years), and seven pigs (<6 months). Hydration of all samples was maintained using Dulbecco’s Modified Eagle Medium (DMEM). Whole lenses were removed from the eye and placed anterior side up in agarose gel before gel hardening where only the posterior half of the lens was contained within the gel. After the gel hardened, the Petri dish was filled with DMEM until the point where the intact lens was fully submerged. AFM was used to image the anterior lens surface in contact mode. An integrated analysis program was used to calculate the interfibrillar spacing, fiber diameter, and surface roughness of the samples. Results The AFM images depict a highly ordered fibrous structure at the surface of the lens capsule in all three species. The interfibrillar spacing for the porcine, cynomolgus monkey, and human lens capsules was 0.68±0.25, 1.80±0.39, and 1.08±0.25 μm, respectively. In the primate, interfibrillar spacing significantly decreased linearly as a function of age. The fiber diameters ranged from 50 to 950 nm. Comparison of the root mean square (RMS) and average deviation demonstrate that the surface of the porcine lens capsule is the smoothest, and that the human and cynomolgus monkey capsules are significantly rougher. Conclusions AFM was successful in providing high-resolution images of the nanostructure of the lens capsule samples. Species-dependent differences were observed in the overall structure and surface roughness. PMID:25814829

Evaluation of 22q11.2 deletion in Cleft Palate patients

PubMed Central

Prabodha, L. B. Lahiru; Dias, Dayanath Kumara; Nanayakkara, B. Ganananda; de Silva, Deepthi C.; Chandrasekharan, N. Vishvanath; Ileyperuma, Isurani

2012-01-01

Background: Cleft palate is the commonest multifactorial epigenetic disorder with a prevalence of 0.43-2.45 per 1000. The objectives of this study were to evaluate the clinical features and identify the 22q11.2 deletion in patients with cleft palate in Sri Lanka. Materials and Methods: Cleft patients attending a Teaching Hospital in Sri Lanka were recruited for this study. The relevant data were obtained from review of case notes, interviews, and examination of patients according to a standard evaluation sheet. Quantitative multiplex polymerase chain reaction (PCR) was performed to identify the 22q11.2 deletion. A gel documentation system (Bio-Doc) was used to quantify the PCR product following electrophoresis on 0.8% agarose gel. Results and Conclusion: There were 162 cleft palate patients of whom 59% were females. A total of 92 cleft palate subjects (56.2%) had other associated clinical features. Dysmorphic features (25.27%) and developmental delays (25.27%) were the commonest medical problems encountered. The cleft was limited to the soft palate in 125 patients, while in 25 patients it involved both the hard and the soft palate. There were seven subjects with bifid uvula and five subjects with submucous cleft palate. None of the patients had 22q11.2 deletion in this study population. A multicentered large population-based study is needed to confirm the results of this study and to develop guidelines on the appropriate use of 22q11.2 deletion testing, which are valid for cleft palate patients in Sri Lanka. PMID:23483617

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

[PCR as a tool in confirming the experimental transmission of Leishmania chagasi to hamsters by Lutzomyia longipalpis (Diptera:Psychodidae)].

PubMed

Cabrera, Olga L; Munstermann, Leonard E; Cárdenas, Rocío; Ferro, Cristina

2003-06-01

The use of PCR (polymerase chain reaction) was evaluated for its effectiveness as a tool in the detection of transmission of Leishmania chagasi to a hamster host, Mesocricetus auratus, by insect vector bite. Two pairs of uninfected and anesthetized hamsters were introduced into cages containing infected females of the typical phlebotomine sand fly vector, Lutzomyia longipalpis. The flies were experimentally infected with Leishmania chagasi and the infection was verified by dissection of subsamples. At 37 and 51 days after exposure to the infected flies, biopsies of each hamster's liver and spleen were subjected to direct histopathological and PCR examination. DNA was extracted with Chelex 100; for PCR amplification, primers specific to Leishmania minicircle DNA were used. PCR product was separated on agarose gels and visualized with UV. A band of approximately 120 base pairs was observed in 3 of the 4 biopsies, corresponding to the expected minicircle size. PCR was the only method that detected presence of the parasite. The results demonstrated that the sensitivity of PCR greatly expedites the confirmation process of a particular phlebotomine species as a vector of leishmaniasis.

Description of polymerase chain reaction and sequencing DNA Mycobacterium tuberculosis from specimen sputum of tuberculosis patients in Medan

NASA Astrophysics Data System (ADS)

Lily; Siregar, Y.; Ilyas, S.

2018-03-01

This study purposed to describe the product Polymerase Chain Reaction (PCR) and sequencing of DNA Mycobacterium (M.) tuberculosis from sputum of tuberculosis (TB) patients in Medan. Sputum was collected from patients that diagnosed with pulmonary TB by a physician. Specimen processed by PCR method of Li et al. and sequencing at Macrogen Laboratory. All of 12 product PCR were showed brightness bands at 126 base pair (bp). These results indicated similarity to the study of Li et al. Sequencing analysis showed the presence of a mutation and non-mutation groups of M. tuberculosis. The reference and outcome berange of the mutation and non-mutation of M. tuberculosis were 56-107, 59-85, 60-120 and 63-94, respectively. The percentage bp difference between the outcome and references for mutation and non-mutation were 3.448-6.569and 3.278-7.428%, respectively. In conclusion, the successful amplification of PCR products in a 1.5% agarose gel electrophoresis where all 12 sputa contained rpoB-positive M. tuberculosis and 0.644% difference was found between the outcome with reference bp of the mutation and non-mutation M. tuberculosis groups.

Applicability of SCAR markers to food genomics: olive oil traceability.

PubMed

Pafundo, Simona; Agrimonti, Caterina; Maestri, Elena; Marmiroli, Nelson

2007-07-25

DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.

Thermodynamic determination of the metal/semiconductor separation of carbon nanotubes using hydrogels.

PubMed

Hirano, Atsushi; Tanaka, Takeshi; Kataura, Hiromichi

2012-11-27

The metal/semiconductor separation of single-wall carbon nanotubes (SWCNTs) using hydrogels, such as agarose gel and Sephacryl, together with sodium dodecyl sulfate is one of the most successful techniques necessary for industrial applications. Despite recent improvements in the technique, little is known about the separation mechanism. Here, we show that SWCNTs are reversibly adsorbed onto hydrogels in the presence of sodium dodecyl sulfate. The results enabled us to examine the thermodynamics of the adsorption reaction and thereby elucidate the separation mechanism. The adsorbability of SWCNTs onto the hydrogels was described by the standard Gibbs free energy for the adsorption, as well as the area of the hydrogels allowing the adsorption. We demonstrated, for the first time, that the free energy of adsorption for semiconducting SWCNTs was 0-12 kJ/mol lower than that for metallic SWCNTs in the temperature range of 290-320 K (e.g., ca. -4 kJ/mol for the agarose gel and ca. -9 kJ/mol for Sephacryl at 300 K), which permits metal/semiconductor separation. Importantly, the difference in the free energy was attributed to the difference in the enthalpy of adsorption: the enthalpy of adsorption of metallic SWCNTs was ca. 70 kJ/mol higher than that of semiconducting SWCNTs. Thus, the enthalpy of adsorption was found to be an important parameter in the metal/semiconductor separation of SWCNTs using hydrogels. In addition, the thermodynamic parameters depended on the hydrogel type and the surfactant concentration, which is most likely why under certain conditions hydrogels and surfactants produce different separations, e.g., chirality-selective or diameter-selective separation.

Purification and partial characterization of low molecular weight vicilin-like glycoprotein from the seeds of Citrullus lanatus.

PubMed

Yadav, Sushila; Tomar, Anil Kumar; Jithesh, O; Khan, Meraj Alam; Yadav, R N; Srinivasan, A; Singh, Tej P; Yadav, Savita

2011-12-01

The watermelon (Citrullus lanatus) seeds are highly nutritive and contain large amount of proteins and many beneficial minerals such as magnesium, calcium, potassium, iron, phosphorous, zinc etc. In various parts of the world, C. lanatus seed extracts are used to cure cancer, cardiovascular diseases, hypertension, and blood pressure. C. lanatus seed extracts are also used as home remedy for edema and urinary tract problems. In this study, we isolated protein fraction of C. lanatus seeds using various protein separation methods. We successfully purified a low molecular weight vicilin-like glycoprotein using chromatographic methods followed by SDS-PAGE and MALDI-TOF/MS identification. This is the first report of purification of a vicilin like polypeptide from C. lanatus seeds. In next step, we extracted mRNA from immature seeds and reverse transcribed it using suitable forward and reverse primers for purified glycoprotein. The PCR product was analysed on 1% agarose gel and was subsequently sequenced by Dideoxy DNA sequencing method. An amino acid translation of the gene is in agreement with amino acid sequences of the identified peptides.

Antimicrobial activity of peptides derived from olive flounder lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI).

PubMed

Nam, Bo-Hye; Moon, Ji-Young; Park, Eun-Hee; Kim, Young-Ok; Kim, Dong-Gyun; Kong, Hee Jeong; Kim, Woo-Jin; Jee, Young Ju; An, Cheul Min; Park, Nam Gyu; Seo, Jung-Kil

2014-10-17

We describe the antimicrobial function of peptides derived from the C-terminus of the olive flounder LBP BPI precursor protein. The investigated peptides, namely, ofLBP1N, ofLBP2A, ofLBP4N, ofLBP5A, and ofLBP6A, formed α-helical structures, showing significant antimicrobial activity against several Gram-negative bacteria, Gram-positive bacteria, and the yeast Candida albicans, but very limited hemolytic activities. The biological activities of these five analogs were evaluated against biomembranes or artificial membranes for the development of candidate therapeutic agents. Gel retardation studies revealed that peptides bound to DNA and inhibited migration on an agarose gel. In addition, we demonstrated that ofLBP6A inhibited polymerase chain reaction. These results suggested that the ofLBP-derived peptide bactericidal mechanism may be related to the interaction with intracellular components such as DNA or polymerase.

Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking.

PubMed Central

Stancheva, I; Lucchini, R; Koller, T; Sogo, J M

1997-01-01

By using formaldehyde cross-linking of histones to DNA and gel retardation assays we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome- packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify fragments from agarose gels, corresponding to coding, enhancer and promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyse independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of Hpa II and Msp I restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single Hpa II site, located in the promoter region, showed particularly strong correlation with the transcriptional activity. PMID:9108154

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1992-01-01

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression. Images PMID:1475192

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein.

PubMed

Wigle, Jeffrey C; Holwitt, Eric A; Estlack, Larry E; Noojin, Gary D; Saunders, Katharine E; Yakovlev, Valdislav V; Rockwell, Benjamin A

2014-01-01

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

No effect of femtosecond laser pulses on M13, E. coli, DNA, or protein

NASA Astrophysics Data System (ADS)

Wigle, Jeffrey C.; Holwitt, Eric A.; Estlack, Larry E.; Noojin, Gary D.; Saunders, Katharine E.; Yakovlev, Valdislav V.; Rockwell, Benjamin A.

2014-01-01

Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.

Hyaluronic Acid (HA)-Polyethylene glycol (PEG) as injectable hydrogel for intervertebral disc degeneration patients therapy

NASA Astrophysics Data System (ADS)

Putri Kwarta, Cityta; Widiyanti, Prihartini; Siswanto

2017-05-01

Chronic Low Back Pain (CLBP) is one health problem that is often encountered in a community. Inject-able hydrogels are the newest way to restore the disc thickness and hydration caused by disc degeneration by means of minimally invasive surgery. Thus, polymers can be combined to improve the characteristic properties of inject-able hydrogels, leading to use of Hyaluronic Acid (a natural polymer) and Polyethylene glycol (PEG) with Horse Radish Peroxide (HRP) cross linker enzymes. The swelling test results, which approaches were the ideal disc values, were sampled with variation of enzyme concentrations of 0.25 µmol/min/mL. The enzyme concentrations were 33.95%. The degradation test proved that the sample degradation increased along with the decrease of the HRP enzyme concentration. The results of the cytotoxicity assay with MTT assay method showed that all samples resulted in the 90% of living cells are not toxic. In vitro injection, models demonstrated that higher concentration of the enzymes was less state of gel which would rupture when released from the agarose gel. The functional group characterization shows the cross linking bonding in sample with enzyme adding. The conclusion of this study is PEG-HA-HRP enzyme are safe polymer composites which have a potential to be applied as an injectable hydrogel for intervertebral disc degeneration.

Performance characteristics of a reverse transcriptase-polymerase chain reaction assay for the detection of tumor-specific fusion transcripts from archival tissue.

PubMed

Fritsch, Michael K; Bridge, Julia A; Schuster, Amy E; Perlman, Elizabeth J; Argani, Pedram

2003-01-01

Pediatric small round cell tumors still pose tremendous diagnostic problems. In difficult cases, the ability to detect tumor-specific gene fusion transcripts for several of these neoplasms, including Ewing sarcoma/peripheral primitive neuroectodermal tumor (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumor (DSRCT) using reverse transcriptase-polymerase chain reaction (RT-PCR), can be extremely helpful. Few studies to date, however, have systematically examined several different tumor types for the presence of multiple different fusion transcripts in order to determine the specificity and sensitivity of the RT-PCR method, and no study has addressed this issue for formalin-fixed material. The objectives of this study were to address the specificity, sensitivity, and practicality of such an assay applied strictly to formalin-fixed tissue blocks. Our results demonstrate that, for these tumors, the overall sensitivity for detecting each fusion transcript is similar to that reported in the literature for RT-PCR on fresh or formalin-fixed tissues. The specificity of the assay is very high, being essentially 100% for each primer pair when interpreting the results from visual inspection of agarose gels. However, when these same agarose gels were examined using Southern blotting, a small number of tumors also yielded reproducibly detectable weak signals for unexpected fusion products, in addition to a strong signal for the expected fusion product. Fluorescence in situ hybridization (FISH) studies in one such case indicated that a rearrangement that would account for the unexpected fusion was not present, while another case was equivocal. The overall specificity for each primer pair used in this assay ranged from 94 to 100%. Therefore, RT-PCR using formalin-fixed paraffin-embedded tissue sections can be used to detect chimeric transcripts as a reliable, highly sensitive, and highly specific diagnostic assay. However, we strongly suggest that the final interpretation of the results from this assay be viewed in light of the other features of the case, including clinical history, histology, and immunohistochemistry, by the diagnostic pathologist. Additional studies such as FISH may be useful in clarifying the nature of equivocal or unexpected results.

A multiscale MD-FE model of diffusion in composite media with internal surface interaction based on numerical homogenization procedure.

PubMed

Kojic, M; Milosevic, M; Kojic, N; Kim, K; Ferrari, M; Ziemys, A

2014-02-01

Mass transport by diffusion within composite materials may depend not only on internal microstructural geometry, but also on the chemical interactions between the transported substance and the material of the microstructure. Retrospectively, there is a gap in methods and theory to connect material microstructure properties with macroscale continuum diffusion characteristics. Here we present a new hierarchical multiscale model for diffusion within composite materials that couples material microstructural geometry and interactions between diffusing particles and the material matrix. This model, which bridges molecular dynamics (MD) and the finite element (FE) method, is employed to construct a continuum diffusion model based on a novel numerical homogenization procedure. The procedure is general and robust for evaluating constitutive material parameters of the continuum model. These parameters include the traditional bulk diffusion coefficients and, additionally, the distances from the solid surface accounting for surface interaction effects. We implemented our models to glucose diffusion through the following two geometrical/material configurations: tightly packed silica nanospheres, and a complex fibrous structure surrounding nanospheres. Then, rhodamine 6G diffusion analysis through an aga-rose gel network was performed, followed by a model validation using our experimental results. The microstructural model, numerical homogenization and continuum model offer a new platform for modeling and predicting mass diffusion through complex biological environment and within composite materials that are used in a wide range of applications, like drug delivery and nanoporous catalysts.

A multiscale MD–FE model of diffusion in composite media with internal surface interaction based on numerical homogenization procedure

PubMed Central

Kojic, M.; Milosevic, M.; Kojic, N.; Kim, K.; Ferrari, M.; Ziemys, A.

2014-01-01

Mass transport by diffusion within composite materials may depend not only on internal microstructural geometry, but also on the chemical interactions between the transported substance and the material of the microstructure. Retrospectively, there is a gap in methods and theory to connect material microstructure properties with macroscale continuum diffusion characteristics. Here we present a new hierarchical multiscale model for diffusion within composite materials that couples material microstructural geometry and interactions between diffusing particles and the material matrix. This model, which bridges molecular dynamics (MD) and the finite element (FE) method, is employed to construct a continuum diffusion model based on a novel numerical homogenization procedure. The procedure is general and robust for evaluating constitutive material parameters of the continuum model. These parameters include the traditional bulk diffusion coefficients and, additionally, the distances from the solid surface accounting for surface interaction effects. We implemented our models to glucose diffusion through the following two geometrical/material configurations: tightly packed silica nanospheres, and a complex fibrous structure surrounding nanospheres. Then, rhodamine 6G diffusion analysis through an aga-rose gel network was performed, followed by a model validation using our experimental results. The microstructural model, numerical homogenization and continuum model offer a new platform for modeling and predicting mass diffusion through complex biological environment and within composite materials that are used in a wide range of applications, like drug delivery and nanoporous catalysts. PMID:24578582

Isolation and evaluation of dental pulp stem cells from teeth with advanced periodontal disease.

PubMed

Derakhshani, Ali; Raoof, Maryam; Dabiri, Shahriar; Farsinejad, Ali Reza; Gorjestani, Hedayat; Yaghoobi, Mohammad Mehdi; Shokouhinejad, Noushin; Ehsani, Maryam

2015-04-01

Successful isolation of mesenchymal stem cells from waste tissues might be extremely promising for developing stem cell-based therapies. This study aimed to explore whether cells retrieved from teeth extracted due to advanced periodontal disease present mesenchymal stem cell-like properties. Pulp cells were isolated from 15 intact molars and 15 teeth with advanced periodontal disease. Cell proliferation and markers of mesenchymal stem cells were evaluated. Based on the RT-PCR and agarose gel electrophoresis, nucleostemin, Oct-4 and jmj2c, but not Nanog, were expressed in undifferentiated mesenchymal stem cells of both groups. Interestingly, diseased pulp exhibited higher gene expressions although it was not statistically significant. The average percentage of BrdU positive cells in the diseased group (84.4%, n = 5) was significantly higher than that of the control group (65.4%, n = 5) (t-test, P = 0.001). Our results indicate the successful isolation of mesenchymal stem cells from the pulp tissue of hopeless periodontally involved teeth.

A polymorphism in the bovine gamma-S-crystallin gene revealed by allele-specific amplification.

PubMed

Kemp, S J; Maillard, J C; Teale, A J

1993-04-01

A polymorphism was detected in the 3' untranslated region of the bovine gamma-S-crystallin gene by direct sequencing of polymerase chain reaction (PCR) products from genomic DNA of an N'Dama bull and a Boran cow. A set of three PCR primers was designed to detect this difference and thus give allele-specific amplification. The two allele-specific primers differ in length by 20 nucleotides so that the allelic products may be distinguished by simple agarose gel electrophoresis following a single PCR reaction. This provides a simple and rapid assay for this polymorphism.

Functionalized Agarose Self-Healing Ionogels Suitable for Supercapacitors.

PubMed

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.

A new prospect in magnetic nanoparticle-based cancer therapy: Taking credit from mathematical tissue-mimicking phantom brain models.

PubMed

Saeedi, Mostafa; Vahidi, Omid; Goodarzi, Vahabodin; Saeb, Mohammad Reza; Izadi, Leila; Mozafari, Masoud

2017-11-01

Distribution patterns/performance of magnetic nanoparticles (MNPs) was visualized by computer simulation and experimental validation on agarose gel tissue-mimicking phantom (AGTMP) models. The geometry of a complex three-dimensional mathematical phantom model of a cancer tumor was examined by tomography imaging. The capability of mathematical model to predict distribution patterns/performance in AGTMP model was captured. The temperature profile vs. hyperthermia duration was obtained by solving bio-heat equations for four different MNPs distribution patterns and correlated with cell death rate. The outcomes indicated that bio-heat model was able to predict temperature profile throughout the tissue model with a reasonable precision, to be applied for complex tissue geometries. The simulation results on the cancer tumor model shed light on the effectiveness of the studied parameters. Copyright © 2017 Elsevier Inc. All rights reserved.

Hairpin-shaped tetranuclear palladium(II) complex: synthesis, crystal structure, DNA binding and cytotoxicity activity studies.

PubMed

Gao, En-Jun; Wang, Ke-Hua; Zhu, Ming-Chang; Liu, Lei

2010-07-01

A novel tetranuclear palladium(II) complex [Pd(4)(phen)(4) (micro-pydc)(4)].10H(2)O (phen = 1,10-phenanthroline, pydc = pyridine-3,4-dicarboxylate) has been synthesized and characterized. In the tetranuclear complex, two pairs of dipalladated [Pd(phen)] moieties are bridged together by four pydc, presenting a hairpin molecular shape. The binding of the title complex with fish sperm DNA (FS-DNA) has been investigated by UV spectrum and fluorescence spectrum. All the results indicate that the complex bind to DNA in an intercalative mode and considerating the molecular shape and size, the dipalladated phenanthroline moieties bisintercalate to the base pairs of DNA. Agarose gel electrophoresis assay demonstrates the ability of the complex to cleave the pBR322 plasmid DNA. Cytotoxic activity studies show the complex exhibited good cytotoxic activity against four different cancer cell lines. Crown Copyright (c) 2010. Published by Elsevier Masson SAS. All rights reserved.

TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.

PubMed

Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi

2014-04-01

Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

PubMed

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.

Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments

PubMed Central

McElfresh, Cameron; Wong, Lily R.

2015-01-01

Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition. PMID:26070672

Beyond Agar: Gel Substrates with Improved Optical Clarity and Drug Efficiency and Reduced Autofluorescence for Microbial Growth Experiments.

PubMed

Jaeger, Philipp A; McElfresh, Cameron; Wong, Lily R; Ideker, Trey

2015-08-15

Agar, a seaweed extract, has been the standard support matrix for microbial experiments for over a century. Recent developments in high-throughput genetic screens have created a need to reevaluate the suitability of agar for use as colony support, as modern robotic printing systems now routinely spot thousands of colonies within the area of a single microtiter plate. Identifying optimal biophysical, biochemical, and biological properties of the gel support matrix in these extreme experimental conditions is instrumental to achieving the best possible reproducibility and sensitivity. Here we systematically evaluate a range of gelling agents by using the yeast Saccharomyces cerevisiae as a model microbe. We find that carrageenan and Phytagel have superior optical clarity and reduced autofluorescence, crucial for high-resolution imaging and fluorescent reporter screens. Nutrient choice and use of refined Noble agar or pure agarose reduce the effective dose of numerous selective drugs by >50%, potentially enabling large cost savings in genetic screens. Using thousands of mutant yeast strains to compare colony growth between substrates, we found no evidence of significant growth or nutrient biases between gel substrates, indicating that researchers could freely pick and choose the optimal gel for their respective application and experimental condition. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

PubMed Central

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

Mechanisms of action and in vivo antibacterial efficacy assessment of five novel hybrid peptides derived from Indolicidin and Ranalexin against Streptococcus pneumoniae

PubMed Central

Jindal, Hassan Mahmood; Zandi, Keivan; Ong, Kien Chai; Velayuthan, Rukumani Devi; Rasid, Sara Maisha; Samudi Raju, Chandramathi

2017-01-01

Background Antimicrobial peptides (AMPs) are of great potential as novel antibiotics for the treatment of broad spectrum of pathogenic microorganisms including resistant bacteria. In this study, the mechanisms of action and the therapeutic efficacy of the hybrid peptides were examined. Methods TEM, SEM and ATP efflux assay were used to evaluate the effect of hybrid peptides on the integrity of the pneumococcal cell wall/membrane. DNA retardation assay was assessed to measure the impact of hybrid peptides on the migration of genomic DNA through the agarose gel. In vitro synergistic effect was checked using the chequerboard assay. ICR male mice were used to evaluate the in vivo toxicity and antibacterial activity of the hybrid peptides in a standalone form and in combination with ceftriaxone. Results The results obtained from TEM and SEM indicated that the hybrid peptides caused significant morphological alterations in Streptococcus pneumoniae and disrupting the integrity of the cell wall/membrane. The rapid release of ATP from pneumococcal cells after one hour of incubation proposing that the antibacterial action for the hybrid peptides is based on membrane permeabilization and damage. The DNA retardation assay revealed that at 62.5 µg/ml all the hybrid peptides were capable of binding and preventing the pneumococcal genomic DNA from migrating through the agarose gel. In vitro synergy was observed when pneumococcal cells treated with combinations of hybrid peptides with each other and with conventional drugs erythromycin and ceftriaxone. The in vivo therapeutic efficacy results revealed that the hybrid peptide RN7-IN8 at 20 mg/kg could improve the survival rate of pneumococcal bacteremia infected mice, as 50% of the infected mice survived up to seven days post-infection. In vivo antibacterial efficacy of the hybrid peptide RN7-IN8 was signficantly improved when combined with the standard antibiotic ceftriaxone at (20 mg/kg + 20 mg/kg) as 100% of the infected mice survived up to seven days post-infection. Discussion Our results suggest that attacking and breaching the cell wall/membrane is most probably the principal mechanism for the hybrid peptides. In addition, the hybrid peptides could possess another mechanism of action by inhibiting intracellular functions such as DNA synthesis. AMPs could play a great role in combating antibiotic resistance as they can reduce the therapeutic concentrations of standard drugs. PMID:29018620

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

PubMed

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.

Simple agarose micro-confinement array and machine-learning-based classification for analyzing the patterned differentiation of mesenchymal stem cells

PubMed Central

Sato, Asako; Vogel, Viola; Tanaka, Yo

2017-01-01

The geometrical confinement of small cell colonies gives differential cues to cells sitting at the periphery versus the core. To utilize this effect, for example to create spatially graded differentiation patterns of human mesenchymal stem cells (hMSCs) in vitro or to investigate underpinning mechanisms, the confinement needs to be robust for extended time periods. To create highly repeatable micro-fabricated structures for cellular patterning and high-throughput data mining, we employed here a simple casting method to fabricate more than 800 adhesive patches confined by agarose micro-walls. In addition, a machine learning based image processing software was developed (open code) to detect the differentiation patterns of the population of hMSCs automatically. Utilizing the agarose walls, the circular patterns of hMSCs were successfully maintained throughout 15 days of cell culture. After staining lipid droplets and alkaline phosphatase as the markers of adipogenic and osteogenic differentiation, respectively, the mega-pixels of RGB color images of hMSCs were processed by the software on a laptop PC within several minutes. The image analysis successfully showed that hMSCs sitting on the more central versus peripheral sections of the adhesive circles showed adipogenic versus osteogenic differentiation as reported previously, indicating the compatibility of patterned agarose walls to conventional microcontact printing. In addition, we found a considerable fraction of undifferentiated cells which are preferentially located at the peripheral part of the adhesive circles, even in differentiation-inducing culture media. In this study, we thus successfully demonstrated a simple framework for analyzing the patterned differentiation of hMSCs in confined microenvironments, which has a range of applications in biology, including stem cell biology. PMID:28380036

Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method.

PubMed

Maeda, Hiroshi; Kokeguchi, Susumu; Fujimoto, Chiyo; Tanimoto, Ichiro; Yoshizumi, Wakako; Nishimura, Fusanori; Takashiba, Shogo

2005-02-01

A method for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for periodontal pathogen, Porphyromonas gingivalis. A set of six primers was designed by targeting the 16S ribosomal RNA gene. By the detection system, target DNA was amplified and visualized on agarose gel within 30 min under isothermal condition at 64 degrees C with a detection limit of 20 cells of P. gingivalis. Without gel electrophoresis, the LAMP amplicon was directly visualized in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The LAMP reaction was also assessed by white turbidity of magnesium pyrophosphate (a by-product of LAMP) in the tube. Detection limits of these naked-eye inspections were 20 cells and 200 cells, respectively. Although false-positive DNA amplification was observed from more than 10(7) cells of Porphyromonas endodontalis, no amplification was observed in other five related oral pathogens. Further, quantitative detection of P. gingivalis was accomplished by a real-time monitoring of the LAMP reaction using SYBR Green I with linearity over a range of 10(2)-10(6) cells. The real-time LAMP was then applied to clinical samples of dental plaque and demonstrated almost identical results to the conventional real-time PCR with an advantage of rapidity. These findings indicate the potential usefulness of LAMP for detecting and quantifying P. gingivalis, especially in its rapidity and simplicity.

Equivalence-point electromigration acid-base titration via moving neutralization boundary electrophoresis.

PubMed

Yang, Qing; Fan, Liu-Yin; Huang, Shan-Sheng; Zhang, Wei; Cao, Cheng-Xi

2011-04-01

In this paper, we developed a novel method of acid-base titration, viz. the electromigration acid-base titration (EABT), via a moving neutralization boundary (MNR). With HCl and NaOH as the model strong acid and base, respectively, we conducted the experiments on the EABT via the method of moving neutralization boundary for the first time. The experiments revealed that (i) the concentration of agarose gel, the voltage used and the content of background electrolyte (KCl) had evident influence on the boundary movement; (ii) the movement length was a function of the running time under the constant acid and base concentrations; and (iii) there was a good linearity between the length and natural logarithmic concentration of HCl under the optimized conditions, and the linearity could be used to detect the concentration of acid. The experiments further manifested that (i) the RSD values of intra-day and inter-day runs were less than 1.59 and 3.76%, respectively, indicating similar precision and stability in capillary electrophoresis or HPLC; (ii) the indicators with different pK(a) values had no obvious effect on EABT, distinguishing strong influence on the judgment of equivalence-point titration in the classic one; and (iii) the constant equivalence-point titration always existed in the EABT, rather than the classic volumetric analysis. Additionally, the EABT could be put to good use for the determination of actual acid concentrations. The experimental results achieved herein showed a new general guidance for the development of classic volumetric analysis and element (e.g. nitrogen) content analysis in protein chemistry. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integration of acoustic radiation force and optical imaging for blood plasma clot stiffness measurement.

PubMed

Wang, Caroline W; Perez, Matthew J; Helmke, Brian P; Viola, Francesco; Lawrence, Michael B

2015-01-01

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood's transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.

Increased frequency of angiotensin-converting enzyme DD genotype in Saudi overweight and obese patients.

PubMed

El-Hazmi, Mohsen Af; Warsy, Arjumand S

2003-01-01

Several studies have been carried out to investigate the insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) in obese and normal weight individuals, often with contradictory results in different populatios. In some studies, the DD genotype occurs at a high frequency in obesity, while in others no association has been demonstrated. Since obesity and overweight are of frequent occurrence in the Saudi population, we initiated this study to investigate the I/D polymorphism of ACE in obese, overweight and normal weight Saudis. The study group included 457 Saudi males and females. The height and weight were measured and body mass index (BMI) was calculated. Based on the BMI value, the total study population was classified as normal, overweight and obese. A total of 117 obese (BMI >30 (kg/m(2))), 185 overweight (BMI=25-29.9 (kg/m(2))) and 155 normal weight individuals (BMI< (kg/m (2)) were enrolled. Polymerase chain reaction (PCR) and agarose gel electrophoresis were used to study the ACE polymorphism. For the deletion (D) form, a 190 bp and for the insertion (I) form, 490 bp DNA fragment were obtained on 2% agarose gel electrophoresis. The frequency of DD genotype was 76.9% in the obese, and 73.5% in the overweight individuals, compared to 19.66% and 24.86% of the ID genotype in the obese and overweight, respectively. Among the normal weight individuals, the frequency was significantly lower for DD and higher for the ID genotype, i.e., 58.7% for DD and 40% for ID. The allele frequencies in the obese were 0.867 and 0.133 for the D and I alleles, respectively. While in the overweight, the frequencies were 0.859 and 0.141 for these alleles, respectively, compared to 0.787 and 0.213 in the normal weight individuals. The DD genotype and D allele occur at a high frequency in Saudi overweight and obese individuals, and may have some role in fat accumulation by affecting metabolic pathways of fat, thus leading to the development of overweight and obesity.

In Vitro Antioxidant and Antiproliferative Activities of Novel Orange Peel Extract and It's Fractions on Leukemia HL-60 Cells.

PubMed

Diab, Kawthar A E; Shafik, Reham Ezzat; Yasuda, Shin

2015-01-01

In the present work, novel orange peel was extracted with 100%EtOH (ethanol) and fractionated into four fractions namely F1, F2, F3, F4 which were eluted from paper chromatographs using 100%EtOH, 80%EtOH, 50%EtOH and pure water respectively. The crude extract and its four fractions were evaluated for their total polyphenol content (TPC), total flavonoid content (TFC) and radical scavenging activity using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Their cytotoxic activity using WST assay and DNA damage by agarose gel electrophoresis were also evaluated in a human leukemia HL-60 cell line. The findings revealed that F4 had the highest TPC followed by crude extract, F2, F3 and F1. However, the crude extract had the highest TFC followed by F4, F3, F2, and F1. Depending on the values of EC50 and trolox equivalent antioxidant capacity, F4 possessed the strongest antioxidant activity while F1 and F2 displayed weak antioxidant activity. Further, incubation HL-60 cells with extract/fractions for 24h caused an inhibition of cell viability in a concentration- dependent manner. F3 and F4 exhibited a high antiproliferative activity with a narrow range of IC50 values (45.9 - 48.9 μg/ml). Crude extract exhibited the weakest antiproliferative activity with an IC50 value of 314.89 μg/ml. Analysis of DNA fragmentation displayed DNA degradation in the form of a smear-type pattern upon agarose gel after incubation of HL-60 cells with F3 and F4 for 6 h. Overall, F3 and F4 appear to be good sources of phytochemicals with antioxidant and potential anticancer activities.

Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography.

PubMed

Mousavi Hosseini, Kamran; Nasiri, Saleh

2015-01-01

Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin- Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate. PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate. Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purificationfold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). RESULTS of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB; these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml). It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII.

Sex Reversal and Analyses of Possible Involvement of Sex Steroids in Scallop Gonadal Development in Newly Established Organ-Culture Systems.

PubMed

Otani, Ayano; Nakajima, Tadaaki; Okumura, Tomomi; Fujii, Shiro; Tomooka, Yasuhiro

2017-04-01

Many molluscs perform sex reversal, and sex hormones may be involved in the process. In adult scallops, Patinopecten yessoensis, gonadotropin releasing hormone and 17β-estradiol (E 2 ) are involved in male sexual maturation, however, little is known about the effects of E 2 and testosterone (T) on the gonadal differentiation in young scallops. In the present study, scallop gonadal development was analyzed to determine the sex reversal stage in Funka bay, and effects of E 2 and T were examined. In Funka bay, almost all scallops were male at month 12. Scallops equipped with ambiguous gonads were 61.1% at month 16 and disappeared at month 18. Therefore, sex reversal in Funka bay occurs at around month 16. For establishment of organ culture systems for bivalves, Manila clam gonads were cultured in 15% L-15 medium diluted with HBSS containing 10% KSR on agarose gel at 10°C, and the gonads survived for 14 days. Scallop gonads were also able to be cultured in 30% L15 medium diluted with ASW containing 10% KSR on agarose gel for seven days. At mature stage, Foxl2 and Tesk were predominantly expressed in ovary and testis, respectively. When scallop gonads at sex reversal stage were organ-cultured, sex steroid treatment decreased Tesk expression in the majority of scallop gonads at sex reversal stage. However, no obvious change in Foxl2 and Tesk expression was detected in mature gonads in response to either E 2 or T in culture, suggesting sex steroid treatment might affect gonadal development at sex reversal stage.

Infusion-line pressure as a real-time monitor of convection-enhanced delivery in pre-clinical models.

PubMed

Lam, Miu Fei; Foo, Stacy W L; Thomas, Meghan G; Lind, Christopher R P

2014-01-15

Acute convection-enhanced delivery (CED) is a neurosurgical delivery technique that allows for precise and uniform distribution of an infusate to a brain structure. It remains experimental due to difficulties in ensuring successful delivery. Real-time monitoring is able to provide immediate feedback on cannula placement, infusate distribution, and if the infusion is proceeding as planned or is failing due to reflux or catheter obstruction. Pressure gradient is the driving force behind CED, with the infusion pressure being directly proportional to the flow-rate. The aim of this study was to assess the feasibility of using infusion-line pressure profiling to distinguish in real-time between succeeding and failing CED infusions. To do so we delivered cresyl violet dye at 0.5, 1.0 and 2.0 μl/min via CED in vitro using 0.6% agarose gel and in vivo to the rat striatum. Infusions that failed in agarose gel models could only be differentiated late during the procedures. In the rat in vivo model, the infusion-line profiles of obstructed infusions were not distinctive from those of successful infusions. Intraoperative magnetic resonance imaging (MRI) is used for real-time visualisation of cannula placement and infusate distribution. Particularly for animal pre-clinical work, it would be advantageous to supplement MRI with a cheap, accessible technique to monitor infusions and provide a real-time measure of infusion success or failure. Infusion-line pressure monitoring was of limited value in identifying successful CED with small volume infusions, whilst its utility for large volume infusion remains unknown. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Hydrogel Bioprinted Microchannel Networks for Vascularization of Tissue Engineering Constructs

PubMed Central

Bertassoni, Luiz E.; Cecconi, Martina; Manoharan, Vijayan; Nikkhah, Mehdi; Hjortnaes, Jesper; Cristino, Ana Luiza; Barabaschi, Giada; Demarchi, Danilo; Dokmeci, Mehmet R.; Yang, Yunzhi; Khademhosseini, Ali

2014-01-01

Vascularization remains a critical challenge in tissue engineering. The development of vascular networks within densely populated and metabolically functional tissues facilitate transport of nutrients and removal of waste products, thus preserving cellular viability over a long period of time. Despite tremendous progress in fabricating complex tissue constructs in the past few years, approaches for controlled vascularization within hydrogel based engineered tissue constructs have remained limited. Here, we report a three dimensional (3D) micromolding technique utilizing bioprinted agarose template fibers to fabricate microchannel networks with various architectural features within photo cross linkable hydrogel constructs. Using the proposed approach, we were able to successfully embed functional and perfusable microchannels inside methacrylated gelatin (GelMA), star poly (ethylene glycol-co-lactide) acrylate (SPELA), poly (ethylene glycol) dimethacrylate (PEGDMA) and poly (ethylene glycol) diacrylate (PEGDA) hydrogels at different concentrations. In particular, GelMA hydrogels were used as a model to demonstrate the functionality of the fabricated vascular networks in improving mass transport, cellular viability and differentiation within the cell-laden tissue constructs. In addition, successful formation of endothelial monolayers within the fabricated channels was confirmed. Overall, our proposed strategy represents an effective technique for vascularization of hydrogel constructs with useful applications in tissue engineering and organs on a chip. PMID:24860845

A molecular genetic lab to generate inclusive and exclusive forensic evidence: two suspects, a victim, and a bloodstained T-shirt.

PubMed

Smit, Julie; Heath, Daniel D; Walter, Ryan P

2014-01-01

Molecular genetic laboratory exercises can be ineffective due the student's lack of connection to the complex and sequential protocols. In this inquiry-based molecular genetic laboratory exercise, we harness students' fascination with human forensics and provide a real-life scenario using biomolecular techniques to identify "whose blood is on the t-shirt." We use fish blood to create realistic blood stains on clothing and challenge the students to use DNA analyses to clear or implicate suspects. Safety concerns are minimized through the use of fish blood, while maximizing both realism and the likelihood of student success due to fishes' nucleated red blood cells. The goal in designing this laboratory exercise was to create a feasible protocol for large (over 300 students) second year university courses. During two 3 hour laboratory sessions, students learn and apply clean/sterile technique, DNA extraction, polymerase chain reaction, restriction fragment length polymorphisms, and agarose gel electrophoresis. The students also learn to interpret the resulting gel bands in terms of inclusive or exclusive evidence. Students have consistently ranked this lab as their favorite of five taken as part of a second year Genetics course. Copyright © 2013 by The International Union of Biochemistry and Molecular Biology.

Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

PubMed

Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

2015-12-28

Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.

PubMed

Even-Chen, S; Barenholz, Y

2000-12-20

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.

Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma.

PubMed

Warton, Kristina; Yuwono, Nicole L; Cowley, Mark J; McCabe, Mark J; So, Alwin; Ford, Caroline E

2017-10-01

Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution. While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.

Multifunctional Magnetic and Upconverting Nanobeads as Dual Modal Imaging Tools.

PubMed

Materia, Maria Elena; Pernia Leal, Manuel; Scotto, Marco; Balakrishnan, Preethi Bala; Kumar Avugadda, Sahitya; García-Martín, María L; Cohen, Bruce E; Chan, Emory M; Pellegrino, Teresa

2017-11-15

We report the fabrication of aqueous multimodal imaging nanocomposites based on superparamagnetic nanoparticles (MNPs) and two different sizes of photoluminescent upconverting nanoparticles (UCNPs). The controlled and simultaneous incorporation of both types of nanoparticles (NPs) was obtained by controlling the solvent composition and the addition rate of the destabilizing solvent. The magnetic properties of the MNPs remained unaltered after their encapsulation into the polymeric beads as shown by the T2 relaxivity measurements. The UCNPs maintain photoluminescent properties even when embedded with the MNPs into the polymer bead. Moreover, the light emitted by the magnetic and upconverting nanobeads (MUCNBs) under NIR excitation (λ exc = 980 nm) was clearly observed through different thicknesses of agarose gel or through a mouse skin layer. The comparison with magnetic and luminescent nanobeads based on red-emitting quantum dots (QDs) demonstrated that while the QD-based beads show significant autofluorescence background from the skin, the signal obtained by the MUCNBs allows a decrease in this background. In summary, these results indicate that MUCNBs are good magnetic and optical probes for in vivo multimodal imaging sensors.

Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.).

PubMed

Rodrigues, Simone M; Soares, Virgínia L F; de Oliveira, Tahise M; Gesteira, Abelmon S; Otoni, Wagner C; Costa, Marcio G C

2007-11-01

The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.

Application of multiplex PCR approaches for shark molecular identification: feasibility and applications for fisheries management and conservation in the Eastern Tropical Pacific.

PubMed

Caballero, S; Cardeñosa, D; Soler, G; Hyde, J

2012-03-01

Here we describe the application of new and existing multiplex PCR methodologies for shark species molecular identification. Four multiplex systems (group ID, thresher sharks, hammerhead sharks and miscellaneous shark) were employed with primers previously described and some designed in this study, which allow for species identification after running PCR products through an agarose gel. This system was implemented for samples (bodies and fins) collected from unidentified sharks landed in the port of Buenaventura and from confiscated tissues obtained from illegal fishing around the Malpelo Island Marine Protected Area, Pacific Coast of Colombia. This method has allowed reliable identification, to date, of 407 samples to the genus and/or species levels, most of them (380) identified as the pelagic thresher shark (Alopias pelagicus). Another seven samples were identified as scalloped hammerhead sharks (Sphyrna lewini). This is an easy-to-implement and reliable identification method that could even be used locally to monitor shark captures in the main fishing ports of developed and developing countries. © 2011 Blackwell Publishing Ltd.

Electronic speckle pattern interferometry: a tool for determining diffusion and partition coefficients for proteins in gels.

PubMed

Karlsson, David; Zacchi, Guido; Axelsson, Anders

2002-01-01

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

PubMed Central

Serwer, P; Watson, R H; Hayes, S J

1987-01-01

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA. Images PMID:2822958

Study of the influence of the agarose hydrogel layer thickness on sensitivity of the coated silica microsphere resonator to humidity.

PubMed

Mallik, Arun Kumar; Farrell, Gerald; Wu, Qiang; Semenova, Yuliya

2017-05-10

In this paper, we investigate both theoretically and experimentally the influence of the agarose hydrogel layer thickness on the sensitivity of a proposed relative humidity (RH) sensor based on a silica microsphere resonator coated with agarose hydrogel. The operating principle of the sensor relies on excitation of whispering gallery modes (WGMs) in the coated silica microsphere using the evanescent field of a tapered fiber. A change in the ambient relative humidity is detected by measuring the wavelength shift of the WGMs in the transmission spectrum of the tapered fiber. Using perturbation theory, we analyze the influence of the agarose coating thickness on the sensitivity of the proposed sensor and compare the results of this analysis with experimental findings for different coating layer thicknesses. We demonstrate that an increase in the coating layer thickness initially leads to an increase in the sensitivity to RH and reaches saturation at higher values of the agarose layer thickness. The results of the study are useful for the design and optimization of microsphere sensor parameters to meet a performance specification.

Electrophoretic separation of gold nanoparticles according to bifunctional molecules-induced charge and size.

PubMed

Kim, Jong-Yeob; Kim, Hyung-Bae; Jang, Du-Jeon

2013-03-01

Gold nanospheres modified with bifunctional molecules have been separated and characterized by using agarose gel electrophoresis as well as optical spectroscopy and electron microscopy. The electrophoretic mobility of a gold nanosphere capped with 11-mercaptoundecanoic acid (MUA) has been found to depend on the number of MUA molecules per gold nanosphere, indicating that it increases with the surface charge of the nanoparticle. The extinction spectrum of gold nanospheres capped with MUA at an MUA molecules per gold nanosphere value of 1000 and connected via 1,6-hexanedithiol (HDT) decreases by 33% in magnitude and shifts to the red as largely as 22 nm with the increase of the molar ratio of HDT to MUA (R(HM)). Gold nanospheres capped with MUA and connected via HDT have been separated successfully using gel electrophoresis and characterized by measuring reflectance spectra of discrete electrophoretic bands directly in the gel and by monitoring transmission electron microscope images of gold nanoparticles collected from the discrete bands. Electrophoretic mobility has been found to decrease substantially with the increment of HDT to MUA, indicating that the size of aggregated gold nanoparticles increases with the concentration of HDT. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Macrophages influence a competition of contact guidance and chemotaxis for fibroblast alignment in a fibrin gel coculture assay.

PubMed

Bromberek, B A; Enever, P A J; Shreiber, D I; Caldwell, M D; Tranquillo, R T

2002-05-01

Rat dermal fibroblasts were dispersed initially in the outer shell of a fibrin gel sphere, while the inner core either was devoid of cells or contained peritoneal exudate cells (primarily macrophages), thereby mimicking the inflammatory phase of wound healing. The fibroblasts compacted floating fibrin microspheres over time. In the absence of macrophages, the initial distribution of fibroblasts (only in the shell) induced circumferential alignment of fibrin fibrils via compaction of the shell relative to the core. The aligned fibrils created a contact guidance field, which was manifested by strong circumferential alignment of the fibroblasts. However, in the presence of macrophages, the fibroblasts exhibited more radial alignment despite the simultaneous contact guidance field in the circumferential direction associated with compaction. This was attributed to a chemotactic gradient emanating from the core due to a putative factor(s) released by the macrophages. The presence of a radial chemotactic stimulus was supported by the finding of even greater radial alignment when fibrin microspheres were embedded in an agarose-fibrin gel that abolished compaction and consequently the contact guidance field. Our assay permits the simulation of tissue morphogenetic processes that involve cell guidance phenomena and tractional restructuring of the extracellular matrix.

Purification and characterization of caffeine synthase from tea leaves.

PubMed

Kato, M; Mizuno, K; Fujimura, T; Iwama, M; Irie, M; Crozier, A; Ashihara, H

1999-06-01

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.

DNA damage induced by the direct effect of radiation

NASA Astrophysics Data System (ADS)

Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

2008-10-01

We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

Analysis of PEG oligomers in black gel inks: Discrimination and ink dating.

PubMed

Sun, Qiran; Luo, Yiwen; Xiang, Ping; Yang, Xu; Shen, Min

2017-08-01

Carbon-based black gel inks are common samples in forensic practice of questioned document examination in China, but there are few analytical methods for this type of ink. In this study, a liquid chromatography-.high resolution mass spectrometry (LC-HRMS) method was established for the analysis of PEG oligomers in carbon-based black gel ink entries. The coupled instruments achieve both the identification and quantification of PEG oligomers in ink entries with reproducible results. Twenty carbon-based black gel inks, whose Raman spectra appeared identical, were analyzed using the LC-HRMS method. As a result, the twenty gel inks were classified into four groups according to the distribution of PEG oligomers. Artificially aging of PEG 400 and a gel ink showed that as PEG degraded, the relative amounts of low molecular weight PEG oligomers increased, while those of high molecular weight decreased. The degradation of PEG oligomers in a naturally aged gel ink was consistent with those in the artificially aged samples, but occurred more slowly. This study not only provided a new method for discriminating carbon-based black gel ink entries, but also offered a new approach for studying the relative ink dating of carbon-based black gel ink entries. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

A hemagglutinating variant of Prevotella melaninogenica isolated from the oral cavity.

PubMed

Haraldsson, G; Holbrook, W P

1998-12-01

Strains resembling Prevotella melaninogenica were isolated from healthy subjects and patients with periodontal disease and were identified using: a 5-test phenotypic screen; commercial identification kits; and a 16S rRNA-based polymerase chain reaction (PCR) method. Eleven clinical isolates closely resembling P. melaninogenica, and all from patients with periodontitis, were able to agglutinate erythrocytes. In the electron microscope, hemagglutinating isolates showed fimbria-like structures, that were not seen on non-hemagglutinating isolates. Some strains were further classified with PCR-restriction fragment-length polymorphism (RFLP) of 16S rRNA genes. Amplified 16S rDNA was digested using five different endonucleases, separated with agarose gel electrophoresis, stained and photographed. Photographs were then scanned, digitized and a distance matrix calculated using Dice coefficient, where the presence or absence of a band was used as a character. The distance matrix was plotted as a phenogram. At 70% similarity six clusters were seen. Type strains of separate Prevotella species did not fall into any cluster. Hemagglutinating isolates fell into three clusters: four clustered with the type strains of P. melaninogenica and Prevotella veroralis; four with other P. melaninogenica isolates and two hemagglutinating isolates clustered together Prevotella loescheii. The PCR-RFLP results showed that the hemagglutinating strains did not form a homogenous group inside the Prevotella genus.

Protein Stains to Detect Antigen on Membranes.

PubMed

Dsouza, Anil; Scofield, R Hal

2015-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Protein stains to detect antigen on membranes.

PubMed

D'souza, Anil; Scofield, R Hal

2009-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

PubMed

Hünniger, Tim; Fischer, Christin; Wessels, Hauke; Hoffmann, Antonia; Paschke-Kratzin, Angelika; Haase, Ilka; Fischer, Markus

2015-03-04

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

[Long distance-PCR for detection of factor VIII gene inversion in patients with severe hemophilia A].

PubMed

Ding, Pei-Fang; Sun, Wei-Sheng; Wang, Qin-You; Liu, De-Chun; Zhang, Xue-Qin; Teng, Bin; Shen, Fa-Kui

2003-08-01

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.

Multiplex DNA detection of food allergens on a digital versatile disk.

PubMed

Tortajada-Genaro, Luis A; Santiago-Felipe, Sara; Morais, Sergi; Gabaldón, José Antonio; Puchades, Rosa; Maquieira, Ángel

2012-01-11

The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 μg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.

Anogenital warts contain several distinct species of human papillomavirus.

PubMed Central

Krzyzek, R A; Watts, S L; Anderson, D L; Faras, A J; Pass, F

1980-01-01

Anogenital warts from 26 patients were examined for the presence of human papillomavirus (HPV). Although no whole, intact virus could be identified, varying amounts of nonintegrated HPV DNA were detected in 18 tissue specimens (70%) by employing both an agarose gel-ethidium bromide staining method and the Southern blot hybridization procedure. When hybridization analysis was performed under stringent conditions, six anogenital warts were observed to contain HPV genomic sequences related to either of the cutaneous viruses HPV type 1 (HPV-1) or HPV-2. In 12 tissue samples lacking sequence homology to either HPV-1 or HPV-2 under stringent conditions, HPV-related sequences were detected when the hybridization was performed under less stringent conditions, indicating that an HPV distinct from both HPV-1 and HPV-2 is also associated with these lesions. This anogenital HPV also appeared to be distinct from the other characterized types of HPV. These data indicate that at least three HPVs are associated with anogenital wart disease. Images PMID:6255208

Axial tomography in live cell laser microscopy

NASA Astrophysics Data System (ADS)

Richter, Verena; Bruns, Sarah; Bruns, Thomas; Weber, Petra; Wagner, Michael; Cremer, Christoph; Schneckenburger, Herbert

2017-09-01

Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

Formulation and method for preparing gels comprising hydrous aluminum oxide

DOEpatents

Collins, Jack L.

2014-06-17

Formulations useful for preparing hydrous aluminum oxide gels contain a metal salt including aluminum, an organic base, and a complexing agent. Methods for preparing gels containing hydrous aluminum oxide include heating a formulation to a temperature sufficient to induce gel formation, where the formulation contains a metal salt including aluminum, an organic base, and a complexing agent.

Formulation and method for preparing gels comprising hydrous cerium oxide

DOEpatents

Collins, Jack L; Chi, Anthony

2013-05-07

Formulations useful for preparing hydrous cerium oxide gels contain a metal salt including cerium, an organic base, and a complexing agent. Methods for preparing gels containing hydrous cerium oxide include heating a formulation to a temperature sufficient to induce gel formation, where the formulation contains a metal salt including cerium, an organic base, and a complexing agent.

Surveyor Nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects.

PubMed

Bannwarth, Sylvie; Procaccio, Vincent; Paquis-Flucklinger, Veronique

2005-06-01

Molecular analysis of mitochondrial DNA (mtDNA) is a critical step in diagnosis and genetic counseling of respiratory chain defects. No fast method is currently available for the identification of unknown mtDNA point mutations. We have developed a new strategy based on complete mtDNA PCR amplification followed by digestion with a mismatch-specific DNA endonuclease, Surveyor Nuclease. This enzyme, a member of the CEL nuclease family of plant DNA endonucleases, cleaves double-strand DNA at any mismatch site including base substitutions and small insertions/deletions. After digestion, cleavage products are separated and analyzed by agarose gel electrophoresis. The size of the digestion products indicates the location of the mutation, which is then confirmed and characterized by sequencing. Although this method allows the analysis of 2 kb mtDNA amplicons and the detection of multiple mutations within the same fragment, it does not lead to the identification of homoplasmic base substitutions. Homoplasmic pathogenic mutations have been described. Nevertheless, most homoplasmic base substitutions are neutral polymorphisms while deleterious mutations are typically heteroplasmic. Here, we report that this method can be used to detect mtDNA mutations such as m.3243A>G tRNA(Leu) and m.14709T>C tRNA(Glu) even when they are present at levels as low as 3% in DNA samples derived from patients with respiratory chain defects. Then, we tested five patients suffering from a mitochondrial respiratory chain defect and we identified a variant (m.16189T>C) in two of them, which was previously associated with susceptibility to diabetes and cardiomyopathy. In conclusion, this method can be effectively used to rapidly and completely screen the entire human mitochondrial genome for heteroplasmic mutations and in this context represents an important advance for the diagnosis of mitochondrial diseases.

Cell mechanics, structure, and function are regulated by the stiffness of the three-dimensional microenvironment.

PubMed

Chen, J; Irianto, J; Inamdar, S; Pravincumar, P; Lee, D A; Bader, D L; Knight, M M

2012-09-19

This study adopts a combined computational and experimental approach to determine the mechanical, structural, and metabolic properties of isolated chondrocytes cultured within three-dimensional hydrogels. A series of linear elastic and hyperelastic finite-element models demonstrated that chondrocytes cultured for 24 h in gels for which the relaxation modulus is <5 kPa exhibit a cellular Young's modulus of ∼5 kPa. This is notably greater than that reported for isolated chondrocytes in suspension. The increase in cell modulus occurs over a 24-h period and is associated with an increase in the organization of the cortical actin cytoskeleton, which is known to regulate cell mechanics. However, there was a reduction in chromatin condensation, suggesting that changes in the nucleus mechanics may not be involved. Comparison of cells in 1% and 3% agarose showed that cells in the stiffer gels rapidly develop a higher Young's modulus of ∼20 kPa, sixfold greater than that observed in the softer gels. This was associated with higher levels of actin organization and chromatin condensation, but only after 24 h in culture. Further studies revealed that cells in stiffer gels synthesize less extracellular matrix over a 28-day culture period. Hence, this study demonstrates that the properties of the three-dimensional microenvironment regulate the mechanical, structural, and metabolic properties of living cells. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Direct Visualization of an Impurity Depletion Zone

NASA Technical Reports Server (NTRS)

Chernov, Alex A.; Garcia-Ruiz, Juan Ma; Thomas, Bill R.

2000-01-01

When a crystal incorporates more impurity per unit of its volume than the impurity concentration in solution, the solution in vicinity of the growing crystal is depleted with respect to the impurity I,2. With a stagnant solution, e. g. in microgravity or gels, an impurity depletion zone expands as the crystal grows and results in greater purity in most of the outer portion of the crystal than in the core. Crystallization in gel provides an opportunity to mimic microgravity conditions and visualize the impurity depletion zone. Colorless, transparent apoferritin (M congruent to 450 KDa) crystals were grown in the presence of red holoferritin dimer as a microheterogeneous impurity (M congruent to 900 KDa) within agarose gel by counterdiffusion with Cd(2+) precipitant. Preferential trapping of dimers, (distribution coefficient K = 4 (exp 1,2)) results in weaker red color around the crystals grown in the left tube in the figure as compared to the control middle tube without crystals. The left and the middle tubes contain colored ferritin dimers, the right tube contains colored trimers. The meniscus in the left tube separate gel (below) and liquid solution containing Cd(2+) (above). Similar solutions, though without precipitants, were present on top of the middle and right tube allowing diffusion of dimers and trimers. The area of weaker color intensity around crystals directly demonstrates overlapped impurity depletion zones.

A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2

PubMed Central

Almogren, Adel; Senior, Bernard W; Kerr, Michael A

2007-01-01

A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S-IgA2) was made possible by the development of a new method of purifying S-IgA1, S-IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin-agarose. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of unreduced pure S-IgA2 revealed that, unlike in S-IgA1, a significant proportion of the secretory component was bound non-covalently in S-IgA2. When S-IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the α-chain, was cleaved. This is in contrast to serum IgA1, in which the α-chain was cleaved under the same conditions – direct evidence that secretory component does protect the α-chain from proteolytic cleavage in S-IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S-IgA1 and S-IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S-IgA2 from that in S-IgA1. PMID:17156102

Use of X-Chromosome Inactivation Pattern to Analyze the Clonality of 14 Female Cases of Kaposi Sarcoma.

PubMed

Yuan, Ding; XiuJuan, Wu; Yan, Zhang; JunQin, Liang; Fang, Xiang; Shirong, Yu; Xiaojing, Kang; Yanyan, Feng; Weidong, Wu; Dong, Luo; Qingli, Lu; DeZhi, Zhang; XiongMing, Pu

2015-06-16

Kaposi sarcoma (KS) has features of both neoplastic growth and hyperplastic proliferation. It is the most common tumor seen in patients with HIV infection. Whether KS is a real tumor or a benign hyperplastic disease is not known. Tissues from KS and cutaneous hemangioma lesion DNA were extracted, and then digested with methylation-sensitive restriction endonuclease HpaII. Human androgen receptor gene (HUMARA) was amplified with PCR method and the product was separated on 10% denaturing polyacrylamide gels and stained with ethylene dibromide (EB) to show the polymorphism of HUMARA. Phosphoglycerate kinase (PGK) was amplified and the product was digested by BStXI, agarose gel and EB stained to show the polymorphism of PGK. Finally, we analyzed the clonality of KS. In the 14 patients with KS, heterozygosity of the HUMARA gene was observed in 12 (85.7%) cases. Loss of heterozygosity of HUMARA gene on X-chromosome (without HpaII digestion there were 2 bands, after HpaII digestion there were just 1 of the bands), representing monoclonal origin, was present in 11 cases of Kaposi sarcoma. Heterozygosity of the PGK gene was observed in 5 (35.7%) cases, which all represent monoclonal origin. There was no significant difference according to country, stage, or HIV and HHV-8 (P>0.05). The current findings suggest that Kaposi sarcoma is a clonal neoplasm, not a reactive proliferation.

New primer for specific amplification of the CAG repeat in Huntington disease alleles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bond, C.E.; Hodes, M.E.

1994-09-01

Huntington disease is an autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat near the 5{prime} end of the gene for Huntington disease (IT15). The CAG repeat is flanked by a variable-length CCG repeat that is included in the amplification product obtained with most currently used primer sets and PCR protocols. Inclusion of this adjacent CCG repeat complicates the accurate assessment of CAG repeat length and interferes with the genotype determination of those individuals carrying alleles in the intermediate range between normal and expanded sized. Due to the GC-rich nature of this region, attempts at designingmore » a protocol for amplification of only the CAG repeat have proved unreliable and difficult to execute. We report here the development of a compatible primer set and PCR protocol that yields consistent amplification of the CAG-repeat region. PCR products can be visualized in ethidium bromide-stained agarose gels for rapid screening or in 6% polyacrylamide gels for determination of exact repeat length. This assay produces bands that can be sized accurately, while eliminating most nonspecific products. Fifty-five specimens examined showed consistency with another well-known method, but one that amplifies the CCG repeats as well. The results we obtained also matched the known carrier status of the donors.« less

CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups: synthesis, kinetics, and sequence specificity of reaction with DNA and biological evaluation.

PubMed

Wang, Y; Gupta, R; Huang, L; Lown, J W

1993-12-24

Antitumor agent CC-1065 functional analogues possessing different electron-withdrawing substituents and leaving groups have been synthesized. The extent and the relative rates of DNA cleavage following alkylation by these CPI structures and thermal treatment were determined independently by an ethidium binding assay and by agarose gel electrophoresis experiments. The anticipated preferential covalent binding to adenine sites within the minor groove was confirmed by sequencing determination of selected agents on high-resolution gels. Certain of the synthetic agents, unlike CC-1065, also bind covalently to G sites with weaker intensity. The cytotoxicities of these compounds were also determined against KB cells in vitro. Compounds bearing a bromo or nitro group in the benzene ring and a methylsulfonyl as a leaving group are 10 and 5 times more potent than their unsubstituted counterparts, respectively. Compounds bearing a methylsulfonyl as a leaving group are more potent than those bearing a chlorine.

[Definition of appropriate temperature and storage conditions in the detection of Leishmania DNA with PCR in phlebotomine flies].

PubMed

Cabrera, Olga L; Munsterman, Leonard E; Cárdenas, Rocío; Gutiérrez, Reynaldo; Ferro, Cristina

2002-09-01

For epidemiological studies and control programs of leishmaniasis, taxonomic identification of the etiologic agent of the disease in the insect vector is of critical importance. The implementation of molecular techniques such as the polymerase chain reaction (PCR) has permitted great advances in the efficacy and sensitivity of parasite identification. Previously, these investigations involved labor-intensive dissections and required expert personnel. The present work evaluates the effects of storage methods of phlebotomine samples in the optimization of PCR identification of Leishmania. Females of Lutzomyia longipalpis, from the colony of the Instituto Nacional de Salud, were experimentally infected with Leishmania chagasi (= L. infantum), from the upper Magdalena Valley (Quipile, Cundinamarca, Colombia). The infected insects were preserved in three solutions: 100% ethanol, 70% ethanol, and TE; subsamples of each class were stored at -80 degrees C, -20 degrees C and room temperature. To determine infection rates, samples were dissected and screened microscopically. Chelex 100 was used for extraction of total Leishmania DNA. For PCR amplification, the kinetoplastic minicircle DNA primers OL1 and OL2 of Leishmania were used, and the products were visualized by electrophoresis in 1% agarose gels. For each of the 3 storage conditions, amplifications were successful, producing a approximately 120 base pair product unique to Leishmania. The results demonstrated the advantage of PCR as a routine screening method for detecting infected flies in endemic foci of visceral leishmaniasis. Since storage method did not affect PCR amplification success, the most cost effective method -70% ethanol at room temperature--is the option recommended for storing entomological samples in vector incrimination studies.

A novel inert crystal delivery medium for serial femtosecond crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conrad, Chelsie E.; Basu, Shibom; James, Daniel

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, themore » 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.« less

A novel inert crystal delivery medium for serial femtosecond crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conrad, Chelsie E.; Basu, Shibom; James, Daniel

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, themore » structure of a multi-subunit complex, phycocyanin, was solved to 2.5Å resolution using 300µg 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.« less

A novel inert crystal delivery medium for serial femtosecond crystallography

DOE PAGES

Conrad, Chelsie E.; Basu, Shibom; James, Daniel; ...

2015-06-30

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, themore » 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.« less

Rapid identification of oral isolates of Aggregatibacter actinomycetemcomitans obtained from humans and primates by an ultrafast super convection based polymerase chain reaction

PubMed Central

Karched, M.; Furgang, D.; Sawalha, N.; Fine, D.H.

2017-01-01

Aggregatibacter actinomycetemcomitans is a Gram negative oral bacterium associated with localized aggressive periodontitis (LAP). Detection of A. actinomycetemcomitans in clinical samples is routinely done by PCR. Our aim was to develop a rapid and reliable PCR method that can be used as a chair-side tool to detect A. actinomycetemcomitans in clinical samples. Sensitivity and specificity assessment was performed on buccal and plaque samples obtained from 40 adolescents enrolled in an ongoing LAP study by comparing 20 A. actinomycetemcomitans-positive subjects and 20 who were negative. In a second study, A. actinomycetemcomitans presence was tested in oral samples from eighty-six primates that included rhesus monkeys, chimpanzees, marmosets, tamarins and baboons. All samples were processed for detection of A. actinomycetemcomitans by means of culture, conventional PCR (cPCR) and rapid PCR (rPCR) using a Super Convection based AmpXpress thermal cycler (AlphaHelix, Sweden). For human samples, culture, cPCR and rPCR showed perfect agreement. Using this method A. actinomycetemcomitans was detected in 27 of 32 rhesus monkeys, 4 of 8 chimpanzees and 1 of 34 marmosets. Rapidity of AmpXpress thermal cycler, combined with Ready-To-Go PCR beads (GE Life sciences), a quick DNA extraction kit (Epicentre Biotechnologies, Madison, Wisconsin, USA) and a bufferless fast agarose gel system, made it possible to obtain results on A. actinomycetemcomitans detection within 35 min. We conclude that AmpXpress fast PCR can be conveniently used as a chair-side tool for rapid detection of A. actinomycetemcomitans in clinical samples. PMID:22326236

Method for smoothing the surface of a protective coating

DOEpatents

Sangeeta, D.; Johnson, Curtis Alan; Nelson, Warren Arthur

2001-01-01

A method for smoothing the surface of a ceramic-based protective coating which exhibits roughness is disclosed. The method includes the steps of applying a ceramic-based slurry or gel coating to the protective coating surface; heating the slurry/gel coating to remove volatile material; and then further heating the slurry/gel coating to cure the coating and bond it to the underlying protective coating. The slurry/gel coating is often based on yttria-stabilized zirconia, and precursors of an oxide matrix. Related articles of manufacture are also described.

Formulation and method for preparing gels comprising hydrous hafnium oxide

DOEpatents

Collins, Jack L; Hunt, Rodney D; Montgomery, Frederick C

2013-08-06

Formulations useful for preparing hydrous hafnium oxide gels contain a metal salt including hafnium, an acid, an organic base, and a complexing agent. Methods for preparing gels containing hydrous hafnium oxide include heating a formulation to a temperature sufficient to induce gel formation, where the formulation contains a metal salt including hafnium, an acid, an organic base, and a complexing agent.

Accuracy of iodine removal using dual-energy CT with or without a tin filter: an experimental phantom study.

PubMed

Kawai, Tatsuya; Takeuchi, Mitsuru; Hara, Masaki; Ohashi, Kazuya; Suzuki, Hirochika; Yamada, Kiyotaka; Sugimura, Yuya; Shibamoto, Yuta

2013-10-01

The effects of a tin filter on virtual non-enhanced (VNE) images created by dual-energy CT have not been well evaluated. To compare the accuracy of VNE images between those with and without a tin filter. Two different types of columnar phantoms made of agarose gel were evaluated. Phantom A contained various concentrations of iodine (4.5-1590 HU at 120 kVp). Phantom B consisted of a central component (0, 10, 25, and 40 mgI/cm(3)) and a surrounding component (0, 50, 100, and 200 mgI/cm(3)) with variable iodine concentration. They were scanned by dual-source CT in conventional single-energy mode and dual-energy mode with and without a tin filter. CT values on each gel at the corresponding points were measured and the accuracy of iodine removal was evaluated. On VNE images, the CT number of the gel of Phantom A fell within the range between -15 and +15 HU under 626 and 881 HU at single-energy 120 kVp with and without a tin filter, respectively. With attenuation over these thresholds, iodine concentration of gels was underestimated with the tin filter but overestimated without it. For Phantom B, the mean CT numbers on VNE images in the central gel component surrounded by the gel with iodine concentrations of 0, 50, 100, and 200 mgI/cm(3) were in the range of -19-+6 HU and 21-100 HU with and without the tin filter, respectively. Both with and without a tin filter, iodine removal was accurate under a threshold of iodine concentration. Although a surrounding structure with higher attenuation decreased the accuracy, a tin filter improved the margin of error.

Bio-printing cell-laden Matrigel–agarose constructs

PubMed Central

Fan, Rong; Piou, Marine; Darling, Evan; Cormier, Denis; Sun, Jun; Wan, Jiandi

2017-01-01

3D printing of biological architectures that mimic the structural and functional features of in vivo tissues is of great interest in tissue engineering and the development of transplantable organ constructs. Printable bio-inks that are compatible with cellular activities play critical roles in the process of 3D bio-printing. Although a variety of hydrogels have been used as bio-inks for 3D bio-printing, they inherit poor mechanical properties and/or the lack of essential protein components that compromise their performance. Here, a hybrid Matrigel–agarose hydrogel system has been demonstrated that possesses both desired rheological properties for bio-printing and biocompatibility for long-term (11 days) cell culture. The agarose component in the hybrid hydrogel system enables the maintenance of 3D-printed structures, whereas Matrigel provides essential microenvironments for cell growth. When human intestinal epithelial HCT116 cells are encapsulated in the printed Matrigel–agarose constructs, high cell viability and proper cell spreading morphology are observed. Given that Matrigel is used extensively for 3D cell culturing, the developed 3D-printable Matrigel–agarose system will open a new way to construct Matrigel-based 3D constructs for cell culture and tissue engineering. PMID:27638155

A cell-laden microfluidic hydrogel.

PubMed

Ling, Yibo; Rubin, Jamie; Deng, Yuting; Huang, Catherine; Demirci, Utkan; Karp, Jeffrey M; Khademhosseini, Ali

2007-06-01

The encapsulation of mammalian cells within the bulk material of microfluidic channels may be beneficial for applications ranging from tissue engineering to cell-based diagnostic assays. In this work, we present a technique for fabricating microfluidic channels from cell-laden agarose hydrogels. Using standard soft lithographic techniques, molten agarose was molded against a SU-8 patterned silicon wafer. To generate sealed and water-tight microfluidic channels, the surface of the molded agarose was heated at 71 degrees C for 3 s and sealed to another surface-heated slab of agarose. Channels of different dimensions were generated and it was shown that agarose, though highly porous, is a suitable material for performing microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. While most cells were found to be viable upon initial device fabrication, only those cells near the microfluidic channels remained viable after 3 days, demonstrating the importance of a perfused network of microchannels for delivering nutrients and oxygen to maintain cell viability in large hydrogels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering, diagnostics, and drug screening applications.

Biomolecular Assembly of Gold Nanocrystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Micheel, Christine Marya

2005-05-20

Over the past ten years, methods have been developed to construct discrete nanostructures using nanocrystals and biomolecules. While these frequently consist of gold nanocrystals and DNA, semiconductor nanocrystals as well as antibodies and enzymes have also been used. One example of discrete nanostructures is dimers of gold nanocrystals linked together with complementary DNA. This type of nanostructure is also known as a nanocrystal molecule. Discrete nanostructures of this kind have a number of potential applications, from highly parallel self-assembly of electronics components and rapid read-out of DNA computations to biological imaging and a variety of bioassays. My research focused inmore » three main areas. The first area, the refinement of electrophoresis as a purification and characterization method, included application of agarose gel electrophoresis to the purification of discrete gold nanocrystal/DNA conjugates and nanocrystal molecules, as well as development of a more detailed understanding of the hydrodynamic behavior of these materials in gels. The second area, the development of methods for quantitative analysis of transmission electron microscope data, used computer programs written to find pair correlations as well as higher order correlations. With these programs, it is possible to reliably locate and measure nanocrystal molecules in TEM images. The final area of research explored the use of DNA ligase in the formation of nanocrystal molecules. Synthesis of dimers of gold particles linked with a single strand of DNA possible through the use of DNA ligase opens the possibility for amplification of nanostructures in a manner similar to polymerase chain reaction. These three areas are discussed in the context of the work in the Alivisatos group, as well as the field as a whole.« less

RAPD- and ERIC-Based Typing of Clinical and Environmental Pseudomonas aeruginosa Isolates.

PubMed

Auda, Ibtesam Ghadban; Al-Kadmy, Israa M S; Kareem, Sawsan Mohammed; Lafta, Aliaa Khyuon; A'Affus, Mustafa Hussein Obeid; Khit, Ibrahim Abd Aloahd; Al Kheraif, Abdulaziz Abdullah; Divakar, Darshan Devang; Ramakrishnaiah, Ravikumar

2017-03-01

Pseudomonas aeruginosa is a major cause of nosocomial infection in children and adults, resulting in significant morbidity and mortality due to its ability to acquire drug resistance. The ability of P. aeruginosa in the environment to cause infection in individuals has been reported previously; henceforth, surveillance of the emergence and transmission of P. aeruginosa strains among patients is important for infection control in a clinical setup. Various gene-typing methods have been used for epidemiological typing of P. aeruginosa isolates for the purpose of surveillance. In this work, the suitability and comparability of two typing methods, enterobacterial repetitive intergenic consensus (ERIC)-PCR and random amplification of polymorphic DNA (RAPD)-PCR fingerprinting, were studied to characterize P. aeruginosa strains isolated from clinical and environmental sources. Forty-four clinical and environmental bacterial isolates of P. aeruginosa were collected between October 2015 and January 2016. DNA extraction, ERIC-PCR and RAPD-PCR, agarose gel electrophoresis, and phylogenetic analyses were carried using the unweighted pair-group method with mean. RAPD typing revealed less clonality among clinical isolates, whereas the ERIC method showed greater similarity in comparison with RAPD. Environmental isolates, however, showed greater similarity using RAPD compared with ERIC typing. With only a few exceptions, most clinical isolates were distinct from environmental isolates, irrespective of the typing method. In conclusion, both the RAPD and ERIC typing methods proved to be good tools in understanding clonal diversity. The results also suggest that there is no relationship between clinical and environmental isolates. The absence of clonality among the clinical isolates may indicate that most P. aeruginosa infection cases could be endemic and not epidemic and that endemic infections may be due to nonclonal strains of P. aeruginosa.

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

PubMed

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.

Mechanical signals in plant development: a new method for single cell studies

NASA Technical Reports Server (NTRS)

Lynch, T. M.; Lintilhac, P. M.

1997-01-01

Cell division, which is critical to plant development and morphology, requires the orchestration of hundreds of intracellular processes. In the end, however, cells must make critical decisions, based on a discrete set of mechanical signals such as stress, strain, and shear, to divide in such a way that they will survive the mechanical loads generated by turgor pressure and cell enlargement within the growing tissues. Here we report on a method whereby tobacco protoplasts swirled into a 1.5% agarose entrapment medium will survive and divide. The application of a controlled mechanical load to agarose blocks containing protoplasts orients the primary division plane of the embedded cells. Photoelastic analysis of the agarose entrapment medium can identify the lines of principal stress within the agarose, confirming the hypothesis that cells divide either parallel or perpendicular to the principal stress tensors. The coincidence between the orientation of the new division wall and the orientation of the principal stress tensors suggests that the perception of mechanical stress is a characteristic of individual plant cells. The ability of a cell to determine a shear-free orientation for a new partition wall may be related to the applied load through the deformation of the matrix material. In an isotropic matrix a uniaxial load will produce a rotationally symmetric strain field, which will define a shear-free plane. Where high stress intensities combine with the loading geometry to produce multiaxial loads there will be no axis of rotational symmetry and hence no shear free plane. This suggests that two mechanisms may be orienting the division plane, one a mechanism that works in rotationally symmetrical fields, yielding divisions perpendicular to the compressive tensor, parallel to the long axis of the cell, and one in asymmetric fields, yielding divisions parallel to the short axis of the cell and the compressive tensor.

Trace Hg2+ analysis via quenching of the fluorescence of a CdS-encapsulated DNA nanocomposite.

PubMed

Long, Yunfei; Jiang, Dianlu; Zhu, Xu; Wang, Jianxiu; Zhou, Feimeng

2009-04-01

A novel fluorescent CdS-encapsulated DNA nanocomposite was synthesized via alternate adsorption of Cd(2+) and S(2-) onto the DNA template affixed inside an agarose gel. Confining DNA molecules in the gel matrix reduces the flexibility of the DNA strand, which facilitates the formation of a uniform coating of CdS onto the DNA template. The resultant rod-shaped nanocomposite (40-90 nm in width and 200-300 nm in length) is well dispersed in solution and fluoresces at 330 nm upon excitation at either 228 or 280 nm. The fluorescence is attributed to tiny particles present in the CdS coating. It was found that the fluorescence can be significantly quenched by trace amount of Hg(2+). The high selectivity toward Hg(2+) and the apparent change in the CdS coating upon exposure to Hg(2+) indicate that Hg(2+) has reacted with the CdS coating through formation of the much more insoluble HgS and the bridging S-Hg-S bonds at the surface. The extent of quenching is dependent on the concentration of Hg(2+) in the range of 0.04-13 microM, and a remarkable detection limit (8.6 nM at 30 degrees C and 4.3 nM at 50 degrees C) can be achieved. The feasibility of the method for the analysis of Hg(2+) in a wastewater sample was demonstrated with an excellent relative standard deviation (RSD, 3.4%). The method described herein is simple, selective, and sensitive and obviates the need of extensive sample pretreatment or special instrumentation.

Secondary Crystal Growth on a Cracked Hydrotalcite-Based Film Synthesized by the Sol-Gel Method.

PubMed

Lee, Wooyoung; Lee, Chan Hyun; Lee, Ki Bong

2016-05-02

The sol-gel synthesis method is an attractive technology for the fabrication of ceramic films due to its preparation simplicity and ease of varying the metal composition. However, this technique presents some limitations in relation to the film thickness. Notably, when the film thickness exceeds the critical limit, large tensile stresses occur, resulting in a cracked morphology. In this study, a secondary crystal growth method was introduced as a post-treatment process for Mg/Al hydrotalcite-based films synthesized by the sol-gel method, which typically present a cracked morphology. The cracked hydrotalcite-based film was hydrothermally treated for the secondary growth of hydrotalcite crystals. In the resulting film, hydrotalcite grew with a vertical orientation, and the gaps formed during the sol-gel synthesis were filled with hydrotalcite after the crystal growth. The secondary crystal growth method provides a new solution for cracked ceramic films synthesized by the sol-gel method.

Cyclodextrin-Based Magnetic Nanoparticles for Cancer Therapy

PubMed Central

Jędrzak, Artur; Szutkowski, Kosma; Grześkowiak, Bartosz F.; Markiewicz, Roksana; Jesionowski, Teofil; Jurga, Stefan

2018-01-01

Polydopamine (PDA)-coated magnetic nanoparticles functionalized with mono-6-thio-β-cyclodextrin (SH-βCD) were obtained and characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), Nuclear and Magnetic Resonance Imaging (NMR and MRI), and doxorubicin (DOXO)-loading experiments. The liver cancer cellular internalization of DOXO-loaded nanoparticles was investigated by confocal imaging microscopy. Synthesized nanomaterials bearing a chemotherapeutic drug and a layer of polydopamine capable of absorbing near-infrared light show high performance in the combined chemo- and photothermal therapy (CT-PTT) of liver cancer due to the synergistic effect of both modalities as demonstrated in vitro. Moreover, our material exhibits improved T2 contrast properties, which have been verified using Carr-Purcell-Meiboom-Gill pulse sequence and MRI Spin-Echo imaging of the nanoparticles dispersed in the agarose gel phantoms. Therefore, the presented results cast new light on the preparation of polydopamine-based magnetic theranostic nanomaterials, as well as on the proper methodology for investigation of magnetic nanoparticles in high field MRI experiments. The prepared material is a robust theranostic nanoasystem with great potential in nanomedicine. PMID:29547559

Optically Controlled Signal Amplification for DNA Computation.

PubMed

Prokup, Alexander; Hemphill, James; Liu, Qingyang; Deiters, Alexander

2015-10-16

The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF → ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF → ON or ON → OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification.

A sensitive gel-based global O-glycomics approach reveals high levels of mannosyl glycans in the high mass region of the mouse brain proteome.

PubMed

Breloy, Isabelle; Pacharra, Sandra; Aust, Christina; Hanisch, Franz-Georg

2012-08-01

We developed a gel-based global O-glycomics method applicable for highly complex protein mixtures entrapped in discontinuous gradient gel layers. The protocol is based on in-gel proteolysis with pronase followed by (glyco)peptide elution and off-gel reductive β-elimination. The protocol offers robust performance with sensitivity in the low picomolar range, is compatible with gel-based proteomics, and shows superior performance in global applications in comparison with workflows eliminating glycans in-gel or from electroblotted glycoproteins. By applying this method, we analyzed the O-glycome of human myoblasts and of the mouse brain O-glycoproteome. After semipreparative separation of mouse brain proteins by one-dimensional SDS gel electrophoresis, the O-glycans from proteins in different mass ranges were characterized with a focus on O-mannose-based glycans. The relative proportion of the latter, which generally represent a rare modification, increases to comparatively high levels in the mouse brain proteome in dependence of increasing protein masses.

Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

PubMed

Affi, Mahmoud; Solliec, Camille; Legentilhomme, Patrick; Comiti, Jacques; Legrand, Jack; Jouanneau, Sulivan; Thouand, Gérald

2016-12-01

Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a general purpose model to understand, anticipate, or predict the dysfunction of a biosensor using immobilized bioluminescent bioreporter in a matrix.

Novel mixed ligand complexes of bioactive Schiff base (E)-4-(phenyl (phenylimino) methyl) benzene-1,3-diol and 2-aminophenol/2-aminobenzoic acid: Synthesis, spectral characterization, antimicrobial and nuclease studies

NASA Astrophysics Data System (ADS)

Subbaraj, P.; Ramu, A.; Raman, N.; Dharmaraja, J.

2014-01-01

A novel bidentate Schiff base ligand has been synthesized using 2,4-dihydroxybenzophenone and aniline. Its mixed ligand complexes of MAB type [M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); HA = Schiff base and B = 2-aminophenol/2-aminobenzoic acid] have been synthesized and characterized on the basis of spectral data UV-Vis, IR, 1H NMR, FAB-Mass, EPR, SEM and magnetic studies. All the complexes were soluble in DMF and DMSO. Elemental analysis and molar conductance values indicate that the complexes are non-electrolytes. HA binds with M(II) ions through azomethine and deprotonated phenolic group and B binds through the primary amine group and deprotonated phenolic/carboxylic groups. Using FAB-Mass the cleavage pattern of the ligand (HA) has been established. All the complexes adopt octahedral geometry around the metal ions. It has been confirmed with the help of UV-Vis, IR, 1H NMR and FAB-Mass spectral data. DNA binding activities of the complexes 1d and 2d are studied by UV-Vis spectroscopy and cleavage studies of Schiff base ligand and its complexes 1d and 2d have been by agarose gel electrophoresis method. In vitro biological activities of the free ligand (HA) and their metal complexes (1a-1e and 2a-2e) were screened against few bacteria, Escherichia coli, Staphylococcus saphyphiticus, Staphylococcus aureus, Pseudomonas aeruginosa and fungi Aspergillus niger, Enterobacter species, Candida albicans by well diffusion technique.

Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

PubMed Central

Vandghanooni, Somayeh; Eskandani, Morteza

2011-01-01

Introduction Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods In the current study, we reviewed recent drug delivery researches related to SCGE. Results We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems. PMID:23678412

Polymorphisms in exons 1B and 1C of the type I interleukin-1 receptor gene in patients with endometriosis.

PubMed

D'Amora, Paulo; Sato, Hélio; Girão, Manoel J B C; Silva, Ismael D C G; Schor, Eduardo

2006-09-01

To study possible correlation between the prevalence of polymorphisms in the type I interleukin-1 receptor gene and pelvic endometriosis. Genotypes of 223 women were analyzed: 109 women with surgically and histologically confirmed endometriosis and 114 healthy women. Distributions of two single-base polymorphisms of the human interleukin-1 receptor type I (IL-1RI) gene were evaluated: PstI, due to a C-->T transition in exon 1B and BsrBI a C-->A transition at position 52 in exon 1C. Polymorphisms were detected by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (RFLP) resolved on 3% agarose gels stained with ethidium bromide. Genotypes for PstI polymorphisms did not differ significantly among control and endometriosis (P = 0.058). However, in relation to BsrBI polymorphism, protective risk was observed for the development of endometriosis [OR 0.39-IC 95% (0.2-0.9)]. BsrBI heterozygote genotype (C/A) showed protective effect against endometriosis development.

Measurement of substance P metabolites in rat CNS.

PubMed

Sakurada, T; Le Grevés, P; Stewart, J; Terenius, L

1985-03-01

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.

Evaluation of a Novel Thermal Accelerant for Augmentation of Microwave Energy during Image-guided Tumor Ablation.

PubMed

Park, William Keun Chan; Maxwell, Aaron Wilhelm Palmer; Frank, Victoria Elizabeth; Primmer, Michael Patrick; Collins, Scott Andrew; Baird, Grayson Luderman; Dupuy, Damian Edward

2017-01-01

The primary challenge in thermal ablation of liver tumors (e.g. hepatocellular carcinoma and hepatic colorectal cancer) is the relatively high recurrence rate (~30%) for which incomplete ablation at the periphery of the tumor is the most common reason. In an attempt to overcome this, we have developed a novel thermal accelerant (TA) agent capable of augmenting microwave energy from a distance normally unattainable by a single microwave ablation antenna. This cesium-based block co-polymer compound transforms from a liquid to a gel at body temperature and is intrinsically visible by computed tomography. Using an agarose phantom model, herein we demonstrate that both the rate and magnitude of temperature increase during microwave ablation were significantly greater in the presence of TA when compared with controls. These results suggest robust augmentation of microwave energy, and may translate into larger ablation zone volumes within biologic tissues. Further work using in vivo techniques is necessary to confirm these findings.

Water soluble cationic dextran derivatives containing poly(amidoamine) dendrons for efficient gene delivery.

PubMed

Mai, Kaijin; Zhang, Shanshan; Liang, Bing; Gao, Cong; Du, Wenjun; Zhang, Li-Ming

2015-06-05

To develop new dextran derivatives for efficient gene delivery, the conjugation of poly(amidoamine) dendrons onto biocompatible dextran was carried out by a Cu(I)-catalyzed azide-alkyne cycloaddition, as confirmed by FTIR and (1)H NMR analyses. For resultant dextran conjugates with various generations of poly(amidoamine) dendrons, their buffering capacity and in vitro cytotoxicity were evaluated by acid-base titration and MTT tests, respectively. In particular, their physicochemical characteristics for the complexation with plasmid DNA were investigated by the combined analyses from agarose gel electrophoresis, zeta potential, particle size, transmission electron microscopy and fluorescence emission spectra. Moreover, their complexes with plasmid DNA were studied with respect to their transfection efficiency in human embryonic kidney (HEK293) cell lines. In the case of a higher generation of poly(amidoamine) dendrons, such a dextran conjugate was found to have much lower cytotoxicity and better gene delivery capability when compared to branched polyethylenimine (bPEI, 25kDa), a commonly used gene vector. Copyright © 2015 Elsevier Ltd. All rights reserved.

Roles of beta2-adrenergic receptor gene polymorphisms in a Turkish population with obstructive sleep apnea syndrome or obesity.

PubMed

Gök, I; Celebi, I; Hüseyinoğlu, N; Ozic, C

2014-10-20

We determined the distribution of the Arg16Gly and Gln27Glu polymorphisms of the beta-2 adrenergic receptor gene (ADRB2) in patients with obstructive sleep apnea syndrome as well as a control group in Northeastern Turkey. A total of 52 patients diagnosed with obstructive sleep apnea in a sleep laboratory and 78 control subjects were examined. Peripheral blood samples were taken from patients diagnosed with obstructive sleep apnea by polysomnography. DNA was extracted from blood samples and amplified using polymerase chain reaction. Amplification products were digested with restriction enzymes to investigate gene polymorphisms. Restriction products were extracted from agarose gel electrophoresis and polymorphisms were analyzed using gel images. The Arg16Gly polymorphism was observed in 18 of 52 patients and in 23 of 78 controls. The Gln27Glu polymorphism was observed in 21 of 52 patients and in 28 of 78 controls. In conclusion, there was no correlation among polymorphic frequencies between patient and control groups. Based on the results, these polymorphisms do not contribute to the clinical diagnosis of this syndrome. However, the distribution of Arg16Gly vs Gln27Glu polymorphisms may contribute to obesity in patients with a body mass index greater than 30 (P < 0.05). Different results may be obtained if the parameters of obstructive sleep apnea disease are changed.

Integration of Acoustic Radiation Force and Optical Imaging for Blood Plasma Clot Stiffness Measurement

PubMed Central

Wang, Caroline W.; Perez, Matthew J.; Helmke, Brian P.; Viola, Francesco; Lawrence, Michael B.

2015-01-01

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties. PMID:26042775

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

PubMed

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+ (<20% inhibition). Binding was also inhibited by pharmaceutical iron chelators (desferoxamine or EDTA) or by higher concentrations of weak iron chelators (citrate or silibinin). Investigation of the physiological effects of iron binding by curcumin revealed that curcumin uptake by cultured cells was reduced >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.

Role of carbohydrate in multimeric structure of factor VIII/von Willebrand factor protein.

PubMed Central

Gralnick, H R; Williams, S B; Rick, M E

1983-01-01

The carbohydrate moiety of the factor VIII/von Willebrand (vW) factor protein is important in the expression of vW factor activity and the intravascular survival of the protein. Studies of normal human factor VIII/vW factor protein indicate that there is a requirement of a full complement of penultimate galactose for the maintenance of a normal multimeric structure. Release of penultimate galactose by beta-galactosidase or modification by galactose oxidase results in loss of the largest molecular weight multimers and increased numbers of intermediate and smaller multimers. In contrast, terminal galactose on the factor VIII/vW factor protein does not appear to play a significant role in the maintenance of the multimer organization. The abnormalities in multimeric structure and molecular size were demonstrated by NaDodSO4/polyacrylamide/agarose gel electrophoresis, NaDodSO4/glyoxyl-agarose electrophoresis, and sucrose density ultracentrifugation. These studies indicate that the penultimate galactose plays a role in the maintenance of the largest multimers of the factor VIII/vW factor protein. This may explain why, in some patients with variant forms of vW disease, a carbohydrate abnormality also may affect the multimeric structure of the plasma factor VIII/vW factor protein. Images PMID:6601805

Purification and Characterization of Caffeine Synthase from Tea Leaves1

PubMed Central

Kato, Misako; Mizuno, Kouichi; Fujimura, Tatsuhito; Iwama, Masanori; Irie, Masachika; Crozier, Alan; Ashihara, Hiroshi

1999-01-01

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg−1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5′-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 μm, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases. PMID:10364410

A practical workshop for generating simple DNA fingerprints of plants.

PubMed

Rouzière, A-S; Redman, J E

2011-01-01

Gel electrophoresis DNA fingerprints offer a graphical and visually appealing illumination of the similarities and differences between DNA sequences of different species and individuals. A polymerase chain reaction (PCR) and restriction digest protocol was designed to give high-school students the opportunity to generate simple fingerprints of plants thereby illustrating concepts and techniques in genetics and molecular biology. Three combinations of primers/restriction enzyme targeting chloroplast DNA were sufficient to generate patterns that enabled visual discrimination of plant species. The protocol was tested with a range of common fruit, vegetable, and herb plants that could be easily cultivated and handled in the laboratory. Toxic or hazardous materials such as ethidium bromide and liquid nitrogen were avoided. The protocol was validated as a university outreach workshop targeted at a group of up to 10 high-school students. In a teaching laboratory, students sampled plants, setup the PCR reaction and restriction digest using microliter pipettes, and loaded the digested samples on an agarose gel. The workshop was structured as 2 × 2.5-hour sessions on separate days. The main challenges stemmed from the speed and accuracy of pipetting, especially at the gel loading stage. Feedback from students was largely positive, with the majority reporting that they had both enjoyed and learnt from the experience. Copyright © 2010 Wiley Periodicals, Inc.

Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 1. Video observation.

PubMed

Vergara, Alessandro; Corvino, Ermanno; Sorrentino, Giosué; Piccolo, Chiara; Tortora, Alessandra; Carotenuto, Luigi; Mazzarella, Lelio; Zagari, Adriana

2002-10-01

Single chains of the collagen model polypeptide with sequence (Pro-Pro-Gly)(10), hereafter referred to as (PPG)(10), aggregate to form rod-shaped triple helices. Crystals of (PPG)(10) were grown in the Advanced Protein Crystallization Facility (APCF) both onboard the International Space Station (ISS) and on Earth. The experiments allow the direct comparison of four different crystallization environments for the first time: solution in microgravity ((g), agarose gel in (g, solution on earth, and gel on earth. Both on board and on ground, the crystal growth was monitored by a CCD video camera. The image analysis provided information on the spatial distribution of the crystals, their movement and their growth rate. The analysis of the distribution of crystals reveals that the crystallization process occurs as it does in batch conditions. Slow motions have been observed onboard the ISS. Different to Space-Shuttle experiment, the crystals onboard the ISS moved coherently and followed parallel trajectories. Growth rate and induction time are very similar both in gel and in solution, suggesting that the crystal growth rate is controlled by the kinetics at the interface under the used experimental conditions. These results provide the first data in the crystallogenesis of (PPG)(10), which is a representative member of non-globular, rod-like proteins.

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

PubMed

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.

Evaluation of Phantom-Based Education System for Acupuncture Manipulation

PubMed Central

Lee, In-Seon; Lee, Ye-Seul; Park, Hi-Joon; Lee, Hyejung; Chae, Younbyoung

2015-01-01

Background Although acupuncture manipulation has been regarded as one of the important factors in clinical outcome, it has been difficult to train novice students to become skillful experts due to a lack of adequate educational program and tools. Objectives In the present study, we investigated whether newly developed phantom acupoint tools would be useful to practice-naïve acupuncture students for practicing the three different types of acupuncture manipulation to enhance their skills. Methods We recruited 12 novice students and had them practice acupuncture manipulations on the phantom acupoint (5% agarose gel). We used the Acusensor 2 and compared their acupuncture manipulation techniques, for which the target criteria were depth and time factors, at acupoint LI11 in the human body before and after 10 training sessions. The outcomes were depth of needle insertion, depth error from target criterion, time of rotating, lifting, and thrusting, time error from target criteria and the time ratio. Results After 10 training sessions, the students showed significantly improved outcomes in depth of needle, depth error (rotation, reducing lifting/thrusting), thumb-forward time error, thumb-backward time error (rotation), and lifting time (reinforcing lifting/thrusting). Conclusions The phantom acupoint tool could be useful in a phantom-based education program for acupuncture-manipulation training for students. For advanced education programs for acupuncture manipulation, we will need to collect additional information, such as patient responses, acupoint-specific anatomical characteristics, delicate tissue-like modeling, haptic and visual feedback, and data from an acupuncture practice simulator. PMID:25689598

Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

PubMed

Goubran-Botros, H; Nanak, E; Abdul Nour, J; Birkenmeir, G; Vijayalakshmi, M A

1992-04-24

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.

Selection and characterization of a DNA aptamer to crystal violet.

PubMed

Chen, Yang; Wang, Jine; Zhang, Yajie; Xu, Lijun; Gao, Tian; Wang, Bing; Pei, Renjun

2018-06-13

Aptamers are short single-stranded DNA or RNA, which can be selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). In order to develop novel light-up probes to substitute G-quadruplex (G4), we selected a DNA aptamer for crystal violet (CV), a triphenylmethane light-up dye, by a modified affinity chromatography-based SELEX. The ssDNA pool was first coupled on streptavidin-coated agarose beads through a biotin labeled complementary oligonucleotide, and then the aptamer sequences would be released from agarose beads by CV affinity. This method is simple, straightforward and effective. The aptamer sequence with a low micromolar dissociation constant (Kd) and good specificity was achieved after 11 rounds of selection. The light-up properties of the CV-aptamer were also investigated, and the CV showed dramatic fluorescence enhancement. The CV-aptamer pair could be further used as a novel light-up fluorescent probe to design biosensors.

Analysis of charge transport in gels containing polyoxometallates using methods of different sensitivity to migration.

PubMed

Caban, Karolina; Lewera, Adam; Zukowska, Grazyna Z; Kulesza, Pawel J; Stojek, Zbigniew; Jeffrey, Kenneth R

2006-08-04

Two methods have been used for examination of transport of charge in gels soaked with DMF and containing dissolved polyoxometallates. The first method is based on the analysis of both Cottrellian and steady-state currents and therefore is capable of giving the concentration of the electroactive redox centres and their transport (diffusion-type) coefficient. The second method provides the real diffusion coefficients, i.e. transport coefficients free of migrational influence, for both the substrate and the product of the electrode reaction. Several gels based on poly(methyl methacrylate), with charged (addition of 1-acrylamido-2-methyl-2-propanesulphonic acid to the polymerization mixture) and uncharged chains, have been used in the investigation. The ratio obtained for the diffusion coefficient (second method) and transport coefficient (first method) was smaller for the gels containing charged polymer chains than for the gels with uncharged chains. In part these changes could be explained by the contribution of migration to the transport of polyoxomatallates in the gels. However, the impact of the changes in the polymer-channel capacity at the electrode surface while the electrode process proceeds was also considered. These structural changes should affect differently the methods based on different time domains.

Mineral Precipitation in Porous Media: Laboratory Diffusion Experiments as Analogues for Concretion Formation in Utah and on Mars

NASA Astrophysics Data System (ADS)

Barge, L. M.; Petruska, J.; Potter, S.; Cho, J.; Chan, M.; Nealson, K.

2007-12-01

We present results of laboratory gel diffusion experiments designed to simulate the precipitation of iron minerals in natural systems. Liesegang bands and crystals of various iron minerals were formed in aqueous gels, "mini- concretions" of mineral precipitate were formed in both sand and a sand/agarose mixture, and the formation of hollow mineral spheres was observed in gel precipitation experiments where organics were introduced. These mineral structures are analogous to concretion forms observed in the Navajo Sandstone region of Utah, which have been suggested as terrestrial analogs for the "blueberry" hematite concretions on Mars. Iron mineral precipitates (perhaps with a gel precursor) occur in many forms in the Navajo Sandstone, including "mini- concretions" (solid concretions 1-2 mm in diameter), "rind-like" concretions (hollow spheres of hematite several cm in diameter, surrounding a region of sandstone), and Liesegang banding (banded patterns that form at reaction fronts through diffusion of ions from one reservoir to another). On Mars only small (4-5mm) and mini-concretions (~ 1mm) have been observed; Liesegang bands or large rind-like concretions have not yet been discovered. The varying conditions that give rise to each of these mineral structures in the laboratory indicate that the small, spheroidal types of iron precipitates found in the Utah and Martian environments may be diagnostic of the diffusion medium, presence of organics, and characteristics of fluid in that region.

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum.

PubMed

Molchanova, Valentina; Chikalovets, Irina; Li, Wei; Kobelev, Stanislav; Kozyrevskaya, Svetlana; Bogdanovich, Raisa; Howard, Eric; Belogortseva, Natalia

2005-05-25

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.

Sex determination in goat by amplification of the HMG box using duplex PCR.

PubMed

Shi, Lei; Yue, Wenbin; Ren, Youshe; Lei, Fulin; Zhao, Junxing

2008-05-01

The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.

Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics.

PubMed

Chokkalingam, Venkatachalam; Tel, Jurjen; Wimmers, Florian; Liu, Xin; Semenov, Sergey; Thiele, Julian; Figdor, Carl G; Huck, Wilhelm T S

2013-12-21

Here, we present a platform to detect cytokine (IL-2, IFN-γ, TNF-α) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.

Method of making ionic liquid mediated sol-gel sorbents

DOEpatents

Malik, Abdul; Shearrow, Anne M.

2017-01-31

Ionic liquid (IL)-mediated sol-gel hybrid organic-inorganic materials present enormous potential for effective use in analytical microextraction. One obstacle to materializing this prospect arises from high viscosity of ILs significantly slowing down sol-gel reactions. A method was developed which provides phosphonium-based, pyridinium-based, and imidazolium-based IL-mediated advanced sol-gel organic-inorganic hybrid materials for capillary microextraction. Scanning electron microscopy results demonstrate that ILs can serve as porogenic agents in sol-gel reactions. IL-mediated sol-gel coatings prepared with silanol-terminated polymers provided up to 28 times higher extractions compared to analogous sol-gel coatings prepared without any IL in the sol solution. This study shows that IL-generated porous morphology alone is not enough to provide effective extraction media: careful choice of the organic polymer and the precursor with close sol-gel reactivity must be made to ensure effective chemical bonding of the organic polymer to the created sol-gel material to be able to provide the desired sorbent characteristics.

RF Heating of MRI-Assisted Catheter Steering Coils for Interventional MRI.

PubMed

Settecase, Fabio; Hetts, Steven W; Martin, Alastair J; Roberts, Timothy P L; Bernhardt, Anthony F; Evans, Lee; Malba, Vincent; Saeed, Maythem; Arenson, Ronald L; Kucharzyk, Walter; Wilson, Mark W

2011-03-01

The aim of this study was too assess magnetic resonance imaging (MRI) radiofrequency (RF)-related heating of conductive wire coils used in magnetically steerable endovascular catheters. A three-axis microcoil was fabricated onto a 1.8Fr catheter tip. In vitro testing was performed on a 1.5-T MRI system using an agarose gel-filled vessel phantom, a transmit-receive body RF coil, a steady-state free precession pulse sequence, and a fluoroptic thermometry system. Temperature was measured without simulated blood flow at varying distances from the magnet isocenter and at varying flip angles. Additional experiments were performed with laser-lithographed single-axis microcoil-tipped microcatheters in air and in a saline bath with varied grounding of the microcoil wires. Preliminary in vivo evaluation of RF heating was performed in pigs at 1.5 T with coil-tipped catheters in various positions in the common carotid arteries with steady-state free precession pulse sequence on and off and under physiologic-flow and zero-flow conditions. In tissue-mimicking agarose gel, RF heating resulted in a maximal temperature increase of 0.35°C after 15 minutes of imaging, 15 cm from the magnet isocenter. For a single-axis microcoil, maximal temperature increases were 0.73°C to 1.91°C in air and 0.45°C to 0.55°C in saline. In vivo, delayed contrast-enhanced MRI revealed no evidence of vascular injury, and histopathologic sections from the common carotid arteries confirmed the lack of vascular damage. Microcatheter tip microcoils for endovascular catheter steering in MRI experience minimal RF heating under the conditions tested. These data provide the basis for further in vivo testing of this promising technology for endovascular interventional MRI. Copyright © 2011 AUR. Published by Elsevier Inc. All rights reserved.

A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction

PubMed Central

Aradaib, Imadeldin E; Majid, Ali A

2006-01-01

A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels. Amplification products were not detected when the PCR-based assay was applied to DNA from other blood parasites including Thieleria annulata, Babesia bigemina or nucleic acid free samples. Application of this nPCR-based assay to clinical samples resulted in direct detection of T. evansi from a variety of tissue samples collected from experimentally infected mice and blood from naturally infected camels. The described nPCR-based assay provides a valuable tool to study the epidemiology of T. evansi infection in camels and other susceptible animal populations. PMID:16712737

Validation of microsatellite markers for cytotype discrimination in the model grass Brachypodium distachyon.

PubMed

Giraldo, Patricia; Rodríguez-Quijano, Marta; Vázquez, José F; Carrillo, José M; Benavente, Elena

2012-07-01

Brachypodium distachyon (L.) P. Beauv. (2n = 2x = 10) is a small annual grass species where the existence of three different cytotypes (10, 20, and 30 chromosomes) has long been regarded as a case of autopolyploid series with x = 5. However, it has been demonstrated that the cytotypes assumed to be polyploids represent two separate Brachypodium species recently named as Brachypodium stacei (2n = 2x = 20) and Brachypodium hybridum (2n = 4x = 30). The aim of this study was to find a PCR-based alternative approach that could replace standard cytotyping methods (i.e., chromosome counting and flow cytometry) to characterize each of the three Brachypodium species. We have analyzed with four microsatellite (SSR) markers 83 B. distachyon-type lines from varied locations in Spain, including the Balearic and Canary Islands. Within this set of lines, 64, 4, and 15 had 10, 20, and 30 chromosomes, respectively. The surveyed markers produced cytotype-specific SSR profiles. So, a single amplification product was generated in the diploid samples, with nonoverlapping allelic ranges between the 2n = 10 and 2n = 20 cytotypes, whereas two bands, one in the size range of each of the diploid cytotypes, were amplified in the 2n = 30 lines. Furthermore, the remarkable size difference obtained with the SSR ALB165 allowed the identification of the Brachypodium species by simple agarose gel electrophoresis.

Novel multiregion hybridization assay for the identification of the most prevalent genetic forms of the human immunodeficiency virus type 1 circulating in Portugal.

PubMed

Freitas, Ferdinando B; Esteves, Aida; Piedade, João; Parreira, Ricardo

2013-02-01

The most efficient method for HIV-1 genetic characterization involves full-genome sequencing, but the associated costs, technical features, and low throughput preclude it from being routinely used for the analysis of large numbers of viral strains. Multiregion hybridization assays (MHA) represent an alternative for a consistent genetic analysis of large numbers of viral strains. Classically, MHA rely on the amplification by real-time PCR of several regions scattered along the HIV-1 genome, and on their characterization with clade-specific TaqMan probes (also known as hydrolysis probes). In this context, the aim of our study was the development of a technical variant of an MHA (vMHA(B/G/02)) for genotyping the most prevalent genetic forms of HIV-1 circulating in Portugal. Different sets of primers were designed for universal and clade-specific amplifications of several sections of the viral genome: gag, pol(Pr), pol(RT), vpu, env(gp120), and env(gp41). vMHA(B/G/02) was implemented using a real-time PCR-based approach, with detection dependent on the use of SYBR Green I. As an alternative, a technically less demanding strategy based on conventional PCR and agarose gel analysis of the reaction products was also developed. This method performed with overall good sensitivity and specificity (>91%) when a convenience sample of 45 plasma-derived HIV-1 strains was analyzed. Apart from the detection of subtype B, G, CRF02_AG, and CRF14_BG viruses, several unique B/G recombinant were also detected. Curiously, recombinant viruses including CRF02_AG sequences were not detected in the group of samples analyzed.

A brief review of other notable protein detection methods on acrylamide gels.

PubMed

Kurien, Biji T; Scofield, R Hal

2012-01-01

Several methods have been described to stain proteins analyzed on acrylamide gels. These include ultrasensitive protein detection in one-dimensional and two-dimensional gel electrophoresis using a fluorescent product from the fungus Epicoccum nigrum; a fluorescence-based Coomassie Blue protein staining; visualization of proteins in acrylamide gels using ultraviolet illumination; fluorescence visualization of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution; and increasing the sensitivity four- to sixfold for detecting trace proteins in dye or silver stained polyacrylamide gels using polyethylene glycol 6000. All these methods are reviewed briefly in this chapter.

The pPSU Plasmids for Generating DNA Molecular Weight Markers.

PubMed

Henrici, Ryan C; Pecen, Turner J; Johnston, James L; Tan, Song

2017-05-26

Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders. Sufficient plasmid DNA can be isolated from 100 ml E. coli cultures for the two plasmids to produce 100 bp or 1 kb ladders for 1000 gels. As such, the pPSU1 and pPSU2 plasmids provide reference fragments from 50 to 10000 bp at a fraction of the cost of commercial DNA ladders. The pPSU1 and pPSU2 plasmids are available without licensing restrictions to nonprofit academic users, affording freely available high-quality, low-cost molecular weight standards for molecular biology applications.

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase 1

PubMed Central

Hack, Ethan; Kemp, John D.

1980-01-01

A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:16661312

Comparison of genome size and synthesis of structural proteins of Hirame Rhabdovirus, infectious hematopoietic necrosis virus, and viral hemorrhagic Septicemia virus

USGS Publications Warehouse

Nishizawa, Toyohiko; Yoshimizu, Mamoru; Winton, James R.; Kimura, Takahisa

1991-01-01

Genomic RNA was extracted from purified virions of hirame rhabdovirus (HRV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV). The full-length RNA was analyzed using formaldehyde agarose gel electrophoresis followed by ethidium bromide staining. Compared with an internal RNA size standard, all three viral genomic RNAs appeared to have identical relative mobilities and were estimated to be approximately 10.7 kilobases in length or about 3.7 megadaltons in molecular mass. Structural protein synthesis of HRV, IHNV, and VHSV was studied using cell cultures treated with actinomycin D. At 2 h intervals, proteins were labeled with 35S-methionine, extracted, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The five structural proteins of each of the three viruses appeared in the following order : nucleoprotein (N), matrix protein 1 (M1), matrix protein 2 (M2), glycoprotein (G), and polymerase (L) reflecting both the approximate relative abundance of each protein within infected cells and the gene order within the viral genome.

A comparison between protein crystals grown with vapor diffusion methods in microgravity and protein crystals using a gel liquid-liquid diffusion ground-based method

NASA Technical Reports Server (NTRS)

Miller, Teresa Y.; He, Xiao-Min; Carter, Daniel C.

1992-01-01

Crystals of human serum albumin have been successfully grown in a variety of gels using crystallization conditions otherwise equivalent to those utilized in the popular hanging-drop vapor-equilibrium method. Preliminary comparisons of gel grown crystals with crystals grown by the vapor diffusion method via both ground-based and microgravity methods indicate that crystals superior in size and quality may be grown by limiting solutal convection. Preliminary X-ray diffraction statistics are presented.

Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP).

PubMed

Chen, Changguo; Zhao, Qiangyuan; Guo, Jianwei; Li, Yanjun; Chen, Qiuyuan

2017-08-01

The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg 2+ concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg 2+ concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.

Hybrid microscaffold-based 3D bioprinting of multi-cellular constructs with high compressive strength: A new biofabrication strategy

PubMed Central

Tan, Yu Jun; Tan, Xipeng; Yeong, Wai Yee; Tor, Shu Beng

2016-01-01

A hybrid 3D bioprinting approach using porous microscaffolds and extrusion-based printing method is presented. Bioink constitutes of cell-laden poly(D,L-lactic-co-glycolic acid) (PLGA) porous microspheres with thin encapsulation of agarose-collagen composite hydrogel (AC hydrogel). Highly porous microspheres enable cells to adhere and proliferate before printing. Meanwhile, AC hydrogel allows a smooth delivery of cell-laden microspheres (CLMs), with immediate gelation of construct upon printing on cold build platform. Collagen fibrils were formed in the AC hydrogel during culture at body temperature, improving the cell affinity and spreading compared to pure agarose hydrogel. Cells were proven to proliferate in the bioink and the bioprinted construct. High cell viability up to 14 days was observed. The compressive strength of the bioink is more than 100 times superior to those of pure AC hydrogel. A potential alternative in tissue engineering of tissue replacements and biological models is made possible by combining the advantages of the conventional solid scaffolds with the new 3D bioprinting technology. PMID:27966623

First identification of Sarcocystis hominis in Iranian traditional hamburger.

PubMed

Ahmadi, M Moghaddam; Hajimohammadi, B; Eslami, G; Oryan, A; Yasini Ardakani, S A; Zohourtabar, A; Zare, S

2015-12-01

Zoonotic concerns of cattle sarcocystosis are of importance, because humans are the final host for Sarcocystis hominis. Therefore the meat products containing beef may encompass sarcocysts which endanger food safety. In this study, we described the first report of molecular identification of S. hominis in Iranian traditional hamburgers using PCR-RFLP. Throughout a pilot research that was carried out to setup a molecular approach to identify the Sarcocystis spp., using PCR-RFLP, a sample of raw Iranian traditional hamburger was purchased from a street food seller located in Yazd, central Iran in May 2013. DNA extraction was done, by salting out method; briefly, the sample was lysed with NET buffer. The DNA purification and precipitation was then performed. Amplicon and digestion results were analyzed, using gel agarose electrophoresis. The results showed a PCR product with 926 bp in length after amplification and 376 and 550 bp in length after digestion. This product was identified as S. hominis. To the best of our knowledge, this is the first report of S. hominis infection in Iranian hamburger.

DNA damage by various radiations

NASA Astrophysics Data System (ADS)

Hasegawa, K.; Yoshioka, H.; Yoshioka, H.

1997-01-01

In an attempt to shed light on the influence of tritiated water on DNA we have investigated the post-irradiation damage with a simple plasmid DNA, pBR322 and pUC18. The survival of covalently closed circular (CCC) DNA form was directly followed by agarose gel electrophoresis. The survival percentage of DNA in tritiated water was almost the same as with the irradiation with X-rays at the same absorbed dose. For irradiation with γ-rays, on the other hand, the decay rate was larger than those observed with both tritiated water and X-rays. The percentages of breakage for DNA in tritiated water, X-rays and γ-rays were found to be 34, 38 and 33% at 100 Gy of absorbed dose. The effect of dose rate was not observed for irradiation with tritiated water, X-rays and γ-rays. In order to study protection of DNA against radiation, we investigated the protecting effect of tea catechin which is the main component of (-)-epigallocatechin gallate (EGCg). The protection mechanism for DNA against radiation-induced scission has been studied using ESR spin-trapping method.

A novel bio electro active alginate-aniline tetramer/ agarose scaffold for tissue engineering: synthesis, characterization, drug release and cell culture study.

PubMed

Atoufi, Zhale; Zarrintaj, Payam; Motlagh, Ghodratollah Hashemi; Amiri, Anahita; Bagher, Zohreh; Kamrava, Seyed Kamran

2017-10-01

In this study, synthesis of a novel biocompatible stimuli-responsive conducting hydrogel based on agarose/alginate-aniline tetramer with the capability of a tailored electrically controlled drug-release for neuroregeneration is investigated. First, aniline tetramer is synthesized and grafted onto sodium alginate. Then, this material is added to agarose as an electrical conductivity modifier to obtain Agarose/alginate-aniline tetramer hydrogel. The synthesized materials are characterized by H NMR and FTIR. The hydrogels are prepared with varying content of aniline tetramer and their swelling-deswelling and shape memory behavior is evaluated. The electroactivity and ionic conductivity of hydrogels against temperature is measured. The sample with 10% aniline tetramer (AT10) reveals the highest ionic conductivity. In MTT and SEM assays, AT10 shows the best cell viability and cell proliferation due to its highest ionic conductivity highlighting the fact that electrical stimuli cell signaling. Hydrogels also represent great potentials for passive and electro-stimulated dexamethasone release. These results demonstrate that the newly developed conducting hydrogels are promising materials for neuroregenerative medicine.

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

PubMed

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.

[Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

PubMed

Wang, Ling; Yang, Peng; Li, Shuang-qing; Xu, Shu-hui; Cao, Gui-qun; Zhang, Fa-qiang; Zhang, Mei-xia; Chen, Qing-ying; Xia, Qing-jie; Liu, Kai; Tang, Fang; Zhang, Yuan-zheng

2004-11-01

To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.

Controlling the electrophoretic mobility of single-walled carbon nanotubes: a comparison of theory and experiment.

PubMed

Usrey, Monica L; Nair, Nitish; Agnew, Daniel E; Pina, Cesar F; Strano, Michael S

2007-07-03

The electrophoretic mobilities of single-walled carbon nanotubes (SWNTs) in agarose gels subjected to negatively charged covalent functionalization and noncovalent anionic surfactant adsorption are compared using a simplified hydrodynamic model. Net charges are calculated on the basis of estimated friction coefficients for cylindrical rodlike particles. The effects of functionalization with negatively charged 4-hydroxybenzene diazonium and anionic sodium cholate are quantified and compared with model predictions. The adsorption of Na+ counterions into the nonionic surfactant layer adsorbed on SWNTs (Triton-X-405) is shown to induce a positive charge and reverse the mobility under select conditions. This effect has not been identified or quantified for nanoparticle systems and may be important in the processing of these systems.

rRNA gene restriction patterns as an epidemiological marker in nosocomial outbreaks of Staphylococcus aureus infections.

PubMed

Meugnier, H; Fernandez, M P; Bes, M; Brun, Y; Bornstein, N; Freney, J; Fleurette, J

1993-01-01

rRNA gene restriction patterns (ribotyping) were compared with phage typing, serotyping, enterotoxins and exfoliatin production in the analysis of 26 Staphylococcus aureus strains isolated from two different nosocomial outbreaks. Total DNA was cleaved by EcoRI restriction endonuclease. After agarose gel electrophoresis and Southern transfer, the hybridization of the membranes was done with radiolabelled 16S rRNA gene from Bacillus subtilis inserted into a plasmid vector. Six to 13 fragments were visualized. A core of common fragments was discerned for all strains tested. A full correlation between ribotyping and conventional markers was observed in only one of the outbreaks studied. In both outbreaks, ribotyping proved helpful in characterizing otherwise untypable strains.

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair.

PubMed Central

Hidema, J.; Kumagai, T.; Sutherland, J. C.; Sutherland, B. M.

1997-01-01

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair. PMID:12223592

Black stains in the mixed dentition: a PCR microbiological study of the etiopathogenic bacteria.

PubMed

Saba, C; Solidani, M; Berlutti, F; Vestri, A; Ottolenghi, L; Polimeni, A

2006-01-01

The aim of this work is to emphasize that particular stains on the third cervical of the buccal and lingual surfaces in mixed dentition, called "black stain." Previous research showed the microbiological etiology of this discoloration by chromogen bacterias. Our study shows bacteria spp involved in stains by means of PCR process and electrophoresis gel on the agarose medium. Sample was formed by 100 subject with black stain and 100 control subjects stain-free. A statistical analysis (SPSS 10.0) using X2 was performed in this study. Porphyromonas gingivalis and Prevotella melaninogenica, were not involved in both in black stain subjects and in the control. On the contrary, Actinomyces could be involved in the pigmentation process.

Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment

PubMed Central

Millard, Julie T.; Chuang, Edward; Lucas, James S.; Nagy, Erzsebet E.; Davis, Griffin T.

2013-01-01

A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context. PMID:24363455

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

Agarose drug delivery systems upgraded by surfactants inclusion: critical role of the pore architecture.

PubMed

Marras-Marquez, T; Peña, J; Veiga-Ochoa, M D

2014-03-15

Anionic or non-ionic surfactants have been introduced in agarose-based hydrogels aiming to tailor the release of drugs with different solubility. The release of a hydrophilic model drug, Theophylline, shows the predictable release enhancement that varies depending on the surfactant. However, when the hydrophobic Tolbutamide is considered, an unexpected retarded release is observed. This effect can be explained not only considering the interactions established between the drug loaded micelles and agarose but also to the alteration of the freeze-dried hydrogels microstructure. It has been observed that the modification of the porosity percentage as well as the pore size distribution during the lyophilization plays a critical role in the different phenomena that take place as soon as desiccated hydrogel is rehydrated. The possibility of tailoring the pore architecture as a function of the surfactant nature and percentage can be applied from drug control release to the widespread and growing applications of materials based on hydrogel matrices. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy E; Singh, Anup K; Throckmorton, Daniel J

2015-02-24

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy; Singh, Anup K; Throckmorton, Daniel J

2013-09-03

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hata, Kuniki; Advanced Science Research Center, Japan Atomic Energy Agency, 2-4 Shirakatashirane, Tokai-mura, Naka-gun, Ibaraki 319-1195; Urushibara, Ayumi

Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield ofmore » DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.« less

Novel mixed ligand complexes of bioactive Schiff base (E)-4-(phenyl (phenylimino) methyl) benzene-1,3-diol and 2-aminophenol/2-aminobenzoic acid: synthesis, spectral characterization, antimicrobial and nuclease studies.

PubMed

Subbaraj, P; Ramu, A; Raman, N; Dharmaraja, J

2014-01-03

A novel bidentate Schiff base ligand has been synthesized using 2,4-dihydroxybenzophenone and aniline. Its mixed ligand complexes of MAB type [M=Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); HA=Schiff base and B=2-aminophenol/2-aminobenzoic acid] have been synthesized and characterized on the basis of spectral data UV-Vis, IR, (1)H NMR, FAB-Mass, EPR, SEM and magnetic studies. All the complexes were soluble in DMF and DMSO. Elemental analysis and molar conductance values indicate that the complexes are non-electrolytes. HA binds with M(II) ions through azomethine and deprotonated phenolic group and B binds through the primary amine group and deprotonated phenolic/carboxylic groups. Using FAB-Mass the cleavage pattern of the ligand (HA) has been established. All the complexes adopt octahedral geometry around the metal ions. It has been confirmed with the help of UV-Vis, IR, (1)H NMR and FAB-Mass spectral data. DNA binding activities of the complexes 1d and 2d are studied by UV-Vis spectroscopy and cleavage studies of Schiff base ligand and its complexes 1d and 2d have been by agarose gel electrophoresis method. In vitro biological activities of the free ligand (HA) and their metal complexes (1a-1e and 2a-2e) were screened against few bacteria, Escherichia coli, Staphylococcus saphyphiticus, Staphylococcus aureus, Pseudomonas aeruginosa and fungi Aspergillus niger, Enterobacter species, Candida albicans by well diffusion technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Large-scale single-chirality separation of single-wall carbon nanotubes by simple gel chromatography

PubMed Central

Liu, Huaping; Nishide, Daisuke; Tanaka, Takeshi; Kataura, Hiromichi

2011-01-01

Monostructured single-wall carbon nanotubes (SWCNTs) are important in both scientific research and electronic and biomedical applications; however, the bulk separation of SWCNTs into populations of single-chirality nanotubes remains challenging. Here we report a simple and effective method for the large-scale chirality separation of SWCNTs using a single-surfactant multicolumn gel chromatography method utilizing one surfactant and a series of vertically connected gel columns. This method is based on the structure-dependent interaction strength of SWCNTs with an allyl dextran-based gel. Overloading an SWCNT dispersion on the top column results in the adsorption sites of the column becoming fully occupied by the nanotubes that exhibit the strongest interaction with the gel. The unbound nanotubes flow through to the next column, and the nanotubes with the second strongest interaction with the gel are adsorbed in this stage. In this manner, 13 different (n, m) species were separated. Metallic SWCNTs were finally collected as unbound nanotubes because they exhibited the lowest interaction with the gel. PMID:21556063

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species

PubMed Central

Diekmann, Kerstin; Hodkinson, Trevor R.; Barth, Susanne

2012-01-01

Background and Aims Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne ‘Cashel’. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species. Methods Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species. Key Results All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A8 mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively. Conclusions The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to their ability to monitor genetic diversity within breeding pools, to trace maternal inheritance and to distinguish closely related species. PMID:22419761

Preparation of hydrophobic organic aeorgels

DOEpatents

Baumann, Theodore F.; Satcher, Jr., Joe H.; Gash, Alexander E.

2007-11-06

Synthetic methods for the preparation of hydrophobic organics aerogels. One method involves the sol-gel polymerization of 1,3-dimethoxybenzene or 1,3,5-trimethoxybenzene with formaldehyde in non-aqueous solvents. Using a procedure analogous to the preparation of resorcinol-formaldehyde (RF) aerogels, this approach generates wet gels that can be dried using either supercritical solvent extraction to generate the new organic aerogels or air dried to produce an xerogel. Other methods involve the sol-gel polymerization of 1,3,5 trihydroxy benzene (phloroglucinol) or 1,3 dihydroxy benzene (resorcinol) and various aldehydes in non-aqueous solvents. These methods use a procedure analogous to the one-step base and two-step base/acid catalyzed polycondensation of phloroglucinol and formaldehyde, but the base catalyst used is triethylamine. These methods can be applied to a variety of other sol-gel precursors and solvent systems. These hydrophobic organics aerogels have numerous application potentials in the field of material absorbers and water-proof insulation.

Preparation of hydrophobic organic aeorgels

DOEpatents

Baumann, Theodore F.; Satcher, Jr., Joe H.; Gash, Alexander E.

2004-10-19

Synthetic methods for the preparation of hydrophobic organics aerogels. One method involves the sol-gel polymerization of 1,3-dimethoxybenzene or 1,3,5-trimethoxybenzene with formaldehyde in non-aqueous solvents. Using a procedure analogous to the preparation of resorcinol-formaldehyde (RF) aerogels, this approach generates wet gels that can be dried using either supercritical solvent extraction to generate the new organic aerogels or air dried to produce an xerogel. Other methods involve the sol-gel polymerization of 1,3,5 trihydroxy benzene (phloroglucinol) or 1,3 dihydroxy benzene (resorcinol) and various aldehydes in non-aqueous solvents. These methods use a procedure analogous to the one-step base and two-step base/acid catalyzed polycondensation of phloroglucinol and formaldehyde, but the base catalyst used is triethylamine. These methods can be applied to a variety of other sol-gel precursors and solvent systems. These hydrophobic organics aerogels have numerous application potentials in the field of material absorbers and water-proof insulation.

New electrochemiluminescent biosensors combining polyluminol and an enzymatic matrix.

PubMed

Sassolas, Audrey; Blum, Loïc J; Leca-Bouvier, Béatrice D

2009-06-01

Performant reagentless electrochemiluminescent (ECL) (bio)sensors have been developed using polymeric luminol as the luminophore. The polyluminol film is obtained by cyclic voltammetry (CV) on a screen-printed electrode either in a commonly used H(2)SO(4) medium or under more original near-neutral buffered conditions. ECL responses obtained after performing polymerization either at acidic pH or at pH 6 have been compared. It appears that polyluminol formed in near-neutral medium gives the best responses for hydrogen peroxide detection. Polymerization at pH 6 by cyclic voltammetry gives a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M H(2)O(2) concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H(2)O(2)-producing oxidase. Here, choline oxidase (ChOD) has been chosen as a model enzyme. To develop the biosensor, luminol has been polymerized at pH 6 by CV, and then an enzyme-entrapping matrix has been formed on the above modified working electrode. Different biological (chitosan, agarose, and alginate) and chemical (silica gels, photopolymers, or reticulated matrices) gels have been tested. Best performances have been obtained by associating a ChOD-immobilizing photopolymer with the polyluminol film. In this case, choline can be detected with a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M.

Rapid isolation of mycoviral double-stranded RNA from Botrytis cinerea and Saccharomyces cerevisiae.

PubMed

Castillo, Antonio; Cottet, Luis; Castro, Miguel; Sepúlveda, Felipe

2011-01-25

In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA. A rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing. The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.

Micro- and Nanomechanical Analysis of Articular Cartilage by Indentation-Type Atomic Force Microscopy: Validation with a Gel-Microfiber Composite

PubMed Central

Loparic, Marko; Wirz, Dieter; Daniels, A.U.; Raiteri, Roberto; VanLandingham, Mark R.; Guex, Geraldine; Martin, Ivan; Aebi, Ueli; Stolz, Martin

2010-01-01

Abstract As documented previously, articular cartilage exhibits a scale-dependent dynamic stiffness when probed by indentation-type atomic force microscopy (IT-AFM). In this study, a micrometer-size spherical tip revealed an unimodal stiffness distribution (which we refer to as microstiffness), whereas probing articular cartilage with a nanometer-size pyramidal tip resulted in a bimodal nanostiffness distribution. We concluded that indentation of the cartilage's soft proteoglycan (PG) gel gave rise to the lower nanostiffness peak, whereas deformation of its collagen fibrils yielded the higher nanostiffness peak. To test our hypothesis, we produced a gel-microfiber composite consisting of a chondroitin sulfate-containing agarose gel and a fibrillar poly(ethylene glycol)-terephthalate/poly(butylene)-terephthalate block copolymer. In striking analogy to articular cartilage, the microstiffness distribution of the synthetic composite was unimodal, whereas its nanostiffness exhibited a bimodal distribution. Also, similar to the case with cartilage, addition of the negatively charged chondroitin sulfate rendered the gel-microfiber composite's water content responsive to salt. When the ionic strength of the surrounding buffer solution increased from 0.15 to 2 M NaCl, the cartilage's microstiffness increased by 21%, whereas that of the synthetic biomaterial went up by 31%. When the nanostiffness was measured after the ionic strength was raised by the same amount, the cartilage's lower peak increased by 28%, whereas that of the synthetic biomaterial went up by 34%. Of interest, the higher peak values remained unchanged for both materials. Taken together, these results demonstrate that the nanoscale lower peak is a measure of the soft PG gel, and the nanoscale higher peak measures collagen fibril stiffness. In contrast, the micrometer-scale measurements fail to resolve separate stiffness values for the PG and collagen fibril moieties. Therefore, we propose to use nanostiffness as a new biomarker to analyze structure-function relationships in normal, diseased, and engineered cartilage. PMID:20513418