Safety Assessment of Nitrocellulose and Collodion as Used in Cosmetics.

PubMed

Fiume, Monice M; Bergfeld, Wilma F; Belsito, Donald V; Hill, Ronald A; Klaassen, Curtis D; Liebler, Daniel C; Marks, James G; Shank, Ronald C; Slaga, Thomas J; Snyder, Paul W; Andersen, F Alan

2016-07-01

The Cosmetic Ingredient Review Expert Panel (the Panel) assessed the safety of nitrocellulose and collodion as used in cosmetics, concluding that these ingredients are safe in the present practices of use and concentration in cosmetic formulations. Both ingredients are used almost exclusively in nail product formulations. The maximum concentration of use of nitrocellulose in nail polish and enamels is 22%; for collodion, the maximum reported concentration of use in nail polish and enamel is 14%. The Panel reviewed available animal and clinical data in making its determination of safety. © The Author(s) 2016.

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

Burnham, A K; Fried, L E

PBX 9404 is an early formulation of HMX from which we can learn about the effects of aging in the weapons stockpile. Of particular interest is the presence of 3% nitrocellulose in PBX 9404 as an energetic binder. Nitrocellulose is used pervasively in smokeless gunpowders and was formerly used extensively in the film and art preservation industries. It is well known that nitrocellulose decomposes autocatalytically, and stabilizers, such as the diphenylamine used in PBX 9404, are used to retard its decomposition. Even so, its lifetime is still limited, and the reactions eventually leading to catastrophic autocatalysis are still not understoodmore » well despite years of work. In addition to reducing the available energy in the explosive, degradation of nitrocellulose affects the mechanical properties of the pressed PBX 9404 parts by the associated reduction in molecular weight, which reduces the strength of the binder. A structural formula for a monomer of the nitrocellulose used in PBX 9404 is shown. The initial nitration level is 2.3 of 3.0 possible sites, and they have different reactivities. Degradation of nitrocellulose affects many properties. As an aid in examining historical chemical analysis data, several measures of degradation are given for the simple replacement of a nitro group with a hydrogen. The weight percent of nitrocellulose remaining for an initial concentration of 3% as used in PBX 9404 is also given. Of course, the real degradation reaction is more complicated, including chain scission and crosslinking reactions giving other gas species. During the course of this work, we spent considerable time addressing the question, ''Why is PBX 9404 blue?'' There was actually considerable controversy in the color evolution with aging, and the situation was clarified by Ben Richardson at Pantex. Workers there assured us that PBX 9404 starts with an ivory color. Drying the prill prior to pressing turns it a mottled blue, and well-preserved prill samples retain a blue color decades after formulation. Subsequently, heat and light both send it through a progression of colors from grayish blue, blue-green, green, brown, dirty yellow, mottled tan, and eventually pale tan. The progression is accelerated by oxygen and possibly moisture, as has been shown in several accelerated aging studies. The precise compounds causing the color evolution are uncertain, but they are undoubtedly a progression of quinoidal, nitroso, and nitrated DPA compounds. For example, paranitroso DPA is deep blue. Unfortunately, the location of various nitrated DPAs, which ranged from yellow to orange to red to brown and which were used by Pantex as analytical standards in the 1970s, is not currently known. While the color change is indicative of aging, it is by no means a quantitative measure of the extent of nitrocellulose degradation. Inspection of the literature yielded a variety of kinetic models, and the activation energy typically ranges from 25-35 kcal/mol for T<100 C. This literature qualitatively predicts times for 30% decomposition ranging from a few days at 100 C to 1-2 years at 50 C to 50 years at room temperature. To develop a quantitative model, we used the data of Leider and Seaton, which were collected at conditions most closely matching stockpile conditions for any data set we had available. They used PBX 9404 heated as pressed pellets in closed vessels at temperatures ranging from 50 to 100 C for times up to three years, and they report mass loss, gas yield and composition, and chemical analysis of the residual solid by methods used in stockpile surveillance. Initial kinetic analysis of the weight of remaining nitrocellulose as measured by liquid chromatography and the loss of nitrate esters by a colorimetric technique gave an activation energy of 27 kcal/mol. However, the reaction is complex due to the different stability of the three nitroester positions, and this complexity required either parallel first-order reactions or an nth-order reaction (n=3.6), which is mathematically equivalent to a Gamma distribution of frequency factors, to match the deceleratory character of conversion versus time curves. This model fit the data up to about 60% conversion, when rapid autocatalysis sets in. Next, a sequential first-order model was developed to match the remaining mass of nitrocellulose, its empirical formula, and the yield of various gases. Details of that chemical reaction model are shown. One caveat is in their use is that the solubility of nitrocellulose decreases substantially for nitrogen contents less than 6 wt%. One might suspect solubility as the cause for the rapid increase in nitrocellulose decomposition rate in nitrocellulose at 60% conversion, but that decrease is accompanied by a sharp increase in gas production, so it truly represents a chemical autocatalytic process.« less

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

PubMed

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

A comparison of UV cross-linking and vacuum baking for nucleic acid immobilization and retention

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

Nierzwicki-Bauer, S.A.; Gebhardt, J.S.; Linkkila, L.

The effectiveness of UV cross-linking and in vacuo baking for the immobilization and retention of DNA to various solid supports was investigated. Optimal immobilization treatments for supported and unsupported nitrocellulose and nylon membranes were: UV cross-linking at 254 nm with an exposure of 120 milliJoules/cm{sup 2}, or baking in vacuo for two hours at 80{degrees}C. UV-immobilized nitrocellulose-based membranes showed no increase in sensitivity when compared to baked membranes. An increase in sensitivity was observed for UV-immobilized nylon membranes as compared with baked nylon membranes in some instances, although this varied within lots of the membranes tested. Repeated strippings and heterologousmore » reprobings resulted in loss of target DNA from UV-immobilized nylon membranes as compared to baked nylon membranes. Loss of target DNA from UV-immobilized nitrocellulose-based membranes due to repeated strippings and reprobings was even more pronounced. In vacuo baking of supported and unsupported nitrocellulose and nylon membranes was more effective for immobilization, and more importantly, for retention of target DNA through many reprobings of the same blot.« less

A simple agar plate preparation for effective transfer of Ureaplasma colonies onto nitrocellulose membranes for colony immunoblotting.

PubMed

Zimmerman, Carl-Ulrich R; Stiedl, Thomas; Spergser, Joachim; Rosengarten, Renate

2014-09-01

A simple method for preparing agar plates is presented, which allows an efficient transfer of Ureaplasma colonies to nitrocellulose membranes for subsequent immunological detection. This simple and reproducible procedure was used to demonstrate antigenic variation in the phase-variable mba-locus of Ureaplasma parvum serovar 3. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative determination of enzyme activity in single cells by scanning microelectrode coupled with a nitrocellulose film-covered microreactor by means of a scanning electrochemical microscope.

PubMed

Zhang, Xiaoli; Sun, Fuchan; Peng, Xuewei; Jin, Wenrui

2007-02-01

An electrochemical method for quantitative determination of enzyme activity in single cells was developed by scanning a microelectrode (ME) over a nitrocellulose film-covered microreactor with micropores by means of a scanning electrochemical microscope (SECM). Peroxidase (PO) in neutrophils was chosen as the model system. The microreactor consisted of a microwell with a solution and a nitrocellulose film with micropores. A single cell perforated by digitonin was injected into the microwell. After the perforated cell was lysed and allowed to dry, physiological buffer saline (PBS) containing hydroquinone (H2Q) and H2O2 as substrates of the enzyme-catalyzed reaction was added in the microwell. The microwell containing the extract of the lysed cell and the enzyme substrates was covered with Parafilm to prevent evaporation. The solution in the microwell was incubated for 20 min. In this case, the released PO from the cell converted H2Q into benzoquinone (BQ). Then, the Parafilm was replaced by a nitrocellulose film with micropores to fabricate the microreactor. The microreactor was placed in an electrochemical cell containing PBS, H2Q, and H2O2. After a 10-microm-radius Au ME was inserted into the electrochemical cell and approached down to the microreactor, the ME was scanned along the central line across the microreactor by means of a SECM. The scan curve with a peak was obtained by detecting BQ that diffused out from the microreactor through the micropores on the nitrocellulose film. PO activity could be quantified on the basis of the peak current on the scan curve using a calibration curve. This method had two obvious advantages: no electrode fouling and no oxygen interference.

A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

PubMed

Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

2012-01-01

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

Combustible Cartridge Cases, an Account of the Current Technology and Proposals for Future Development.

DTIC Science & Technology

1986-10-01

mixture of energetic nitrocellulose libres and inert cellulose fibres. Additives, such as polymeric wet strength resins, fillers, waxes and other...produced using inert cellulosic materials, while incorporation of nitrocellulose, a strong oxidiser, results in a ’ombustible’ product. At present...textiles with wet strength resins. The nitrated cellulosic fabric was laminated by winding around a collapsible mandrel which is rotated under pressure

Joint Symposium on Compatibility of Plastics/Materials with Explosives Processing Explosives Held in Albuquerque, New Mexico on 15-17 May 1979.

DTIC Science & Technology

1979-05-01

250 A. S. Tompa REAL-TIME LOW TEMPERATURE NC AND PBX 9404 DECOMPOSITION STUDIES ....................................... 276 ._- Dr. Hermann...the five major unit operations for multi-base cannon propellant; nitrocellulose dehydration , premixing, mixing, extruding and cutting. Throughout the...during facility design, a general process description is presented as follows: Thermal Dehydration Nitrocellulose (NC) slurry is fed to a continuous

High-Content Optical Codes for Protecting Rapid Diagnostic Tests from Counterfeiting.

PubMed

Gökçe, Onur; Mercandetti, Cristina; Delamarche, Emmanuel

2018-06-19

Warnings and reports on counterfeit diagnostic devices are released several times a year by regulators and public health agencies. Unfortunately, mishandling, altering, and counterfeiting point-of-care diagnostics (POCDs) and rapid diagnostic tests (RDTs) is lucrative, relatively simple and can lead to devastating consequences. Here, we demonstrate how to implement optical security codes in silicon- and nitrocellulose-based flow paths for device authentication using a smartphone. The codes are created by inkjet spotting inks directly on nitrocellulose or on micropillars. Codes containing up to 32 elements per mm 2 and 8 colors can encode as many as 10 45 combinations. Codes on silicon micropillars can be erased by setting a continuous flow path across the entire array of code elements or for nitrocellulose by simply wicking a liquid across the code. Static or labile code elements can further be formed on nitrocellulose to create a hidden code using poly(ethylene glycol) (PEG) or glycerol additives to the inks. More advanced codes having a specific deletion sequence can also be created in silicon microfluidic devices using an array of passive routing nodes, which activate in a particular, programmable sequence. Such codes are simple to fabricate, easy to view, and efficient in coding information; they can be ideally used in combination with information on a package to protect diagnostic devices from counterfeiting.

Aviation Ammunition (Selected Chapters)

DTIC Science & Technology

1985-04-01

desensitizers, flash- inhibiting additives, etc. Cellulose nitrates are the base for all nitrocellulose powders . They determine the power of the powder to a...plate, tube, cylinder, etc.) is given to W nitrocellulose powders in the production process. A particle of powder with an established shape and size...used in this case in the following a 4form: .1 (3.4) * The method which has been presented for determining the pressure on * an air shock wave front

ABO grouping of highly-dilute blood by the absorption-elution technique using nitrocellulose beads--application to a casework investigation.

PubMed

Fujitani, N; Matoba, R; Kobayashi, T; Matsuda, H; Yoshida, K; Fukita, K

1991-04-01

This paper reports a homicidal case in which the absorption-elution technique using nitrocellulose beads as immunoadsorbents was successfully applied to ABO grouping from highly-diluted blood. A 21-year-old man was found dead in bed while staying in a hotel. He had multiple wounds over the entire body. By autopsy the cause of death was decided to be traumatic shock. The victim's blood group was A. A bucket filled with faint-colored water was found at the scene. By means of the absorption-elution technique using nitrocellulose beads the water was grouped as B. Later, a 32-year-old man staying in the hotel together with the victim was suspected and arrested. The suspect's blood group was B. He confessed that he had injured himself in the hands with a knife during the struggle and washed them in the water.

Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.

PubMed

Robinson, P A; Anderton, B H; Loviny, T L

1988-04-06

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

Friction and morphology of magnetic tapes in sliding contact with nickel-zinc ferrite

NASA Technical Reports Server (NTRS)

Miyoshi, K.; Buckley, D. H.; Bhushan, B.

1984-01-01

Friction and morphological studies were conducted with magnetic tapes containing a Ni-Zn ferrite hemispherical pin in laboratory air at a relative humidity of 40 percent and at 23 C. The results indicate that the binder plays a significant role in the friction properties, morphology, and microstructure of the tape. Comparisons were made with four binders: nitrocellulose; poly (vinyledene) chloride; cellulose acetate; and hydroxyl-terminated, low molecular weight polyester added to the base polymer, polyester-polyurethane. The coefficient of friction was lowest for the tape with the nitrocellulose binder and increased in the order hydroxylterminated, low molecular weight polyester resin; poly (vinyledene) chloride; and cellulose acetate. The degree of enclosure of the oxide particles by the binder was highest for hydroxyl-terminated, low molecular weight polyester and decreased in the order cellulose acetate, poly (vinyledene) chloride, and nitrocellulose. The nature of deformation of the tape was a factor in controlling friction. The coefficient of friction under elastic contact conditions was considerably lower than under conditions that produced plastic contacts.

Experimental Study on the Fire Properties of Nitrocellulose with Different Structures

PubMed Central

Wei, Ruichao; He, Yaping; Liu, Jiahao; He, Yu; Mi, Wenzhong; Yuen, Richard; Wang, Jian

2017-01-01

In order to ensure the safety of inflammable and explosive chemical substance such as nitrocellulose (NC) mixtures in the process of handing, storage, and usage, it is necessary to obtain the fire properties of NC with different exterior structures. In present study, fire properties of two commonly used nitrocelluloses with soft fiber structure and white chip structure were investigated by scanning electron microscope (SEM) and the ISO 5660 cone calorimeter. Experimental findings revealed that the most important fire properties such as ignition time, mass loss rate and ash content exhibited significant differences between the two structures of NC. Compared with the soft fiber NC, chip NC possesses a lower fire hazard, and its heat release rate intensity (HRRI) is mainly affected by the sample mass. In addition, oxygen consumption (OC) calorimetry method was compared with thermal chemistry (TC) method based on stoichiometry for HRRI calculation. HRRI results of NC with two structures obtained by these two methods showed a good consistency. PMID:28772675

[Better performance of Western blotting: quick vs slow protein transfer, blotting membranes and the visualization methods].

PubMed

Kong, Ling-Quan; Pu, Ying-Hui; Ma, Shi-Kun

2008-01-01

To study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting. The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB). In Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not. Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

Effects of a Near Field Pyroshock on the Performance of a Nitramine Nitrocellulose Propellant

NASA Technical Reports Server (NTRS)

Baca, Arcenio

2016-01-01

The purpose of this study is to investigate the effects of a pyroshock environment on the performance characteristics of a propellant used in pyrotechnic devices such as guillotine cutters. A heritage pressure cartridge assembly which uses a nitramine nitrocellulose propellant with a known performance baseline will be exposed to a near field pyroshock event. The pressure cartridge will then be fired in an ambient closed bomb firing to collect pressure time history. This data will be compared to the baseline data to evaluate the effects of the shock on the performance of the propellant.

Electrospun nanofiber-based thermite textiles and their reactive properties.

PubMed

Yan, Shi; Jian, Guoqiang; Zachariah, Michael R

2012-12-01

In this work, we present a first time fabrication of thermite-based nanofiber mats with a nitrocellulose composite energetic binder to create a new class of energetic 1D nanocomposite. The as prepared thermite based nanofibrous mats were characterized and tested for their burning behavior, and compared with the pure nitrocellulose and nanoaluminum incorporated nanofibers for their combustion performances. Thermite-based nanofibers show enhanced burning rates in combustion tests, which correlate to the mass loading of nanothermite relative to binder in nanofibers. The electrospinning method demonstrates the possibility of avoiding some of the problems associated with melt casting nanometalized propellants.

Combustion-Assisted Photonic Annealing of Printable Graphene Inks via Exothermic Binders.

PubMed

Secor, Ethan B; Gao, Theodore Z; Dos Santos, Manuel H; Wallace, Shay G; Putz, Karl W; Hersam, Mark C

2017-09-06

High-throughput and low-temperature processing of high-performance nanomaterial inks is an important technical challenge for large-area, flexible printed electronics. In this report, we demonstrate nitrocellulose as an exothermic binder for photonic annealing of conductive graphene inks, leveraging the rapid decomposition kinetics and built-in energy of nitrocellulose to enable versatile process integration. This strategy results in superlative electrical properties that are comparable to extended thermal annealing at 350 °C, using a pulsed light process that is compatible with thermally sensitive substrates. The resulting porous microstructure and broad liquid-phase patterning compatibility are exploited for printed graphene microsupercapacitors on paper-based substrates.

Draft genome sequence of Brevibacterium epidermidis EZ-K02 isolated from nitrocellulose-contaminated wastewater environments.

PubMed

Ziganshina, Elvira E; Mohammed, Waleed S; Doijad, Swapnil P; Shagimardanova, Elena I; Gogoleva, Natalia E; Ziganshin, Ayrat M

2018-04-01

Brevibacterium spp. are aerobic, nonbranched, asporogenous, gram-positive, rod-shaped bacteria which may exhibit a rod-coccus cycle when cells get older and can be found in various environments. ​Several Brevibacterium species have industrial importance and are capable of biotransformation of various contaminants. Here we describe the draft genome sequence of Brevibacterium epidermidis EZ-K02 isolated from nitrocellulose-contaminated wastewater environments. The genome comprises 3,885,924 bp, with a G + C content of 64.2%. This whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PDHL00000000.

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Development towards compact nitrocellulose interferometric biochips for dry eye diagnosis based on MMP9, S100A6 and CST4 biomarkers using a Point-of-Care device

NASA Astrophysics Data System (ADS)

Santamaría, Beatriz; Laguna, María. Fe; López-Romero, David; López-Hernandez, A.; Sanza, F. J.; Lavín, A.; Casquel, R.; Maigler, M.; Holgado, M.

2018-02-01

A novel compact optical biochip based on a thin layer-sensing BICELL surface of nitrocellulose is used for in-situ labelfree detection of dry eye disease (DED). In this work the development of a compact biosensor that allows obtaining quantitative diagnosis with a limited volume of sample is reported. The designed sensors can be analyzed with an optical integrated Point-of-Care read-out system based on the "Increase Relative Optical Power" principle which enhances the performance and Limit of Detection. Several proteins involved with dry eye dysfunction have been validated as biomarkers. Presented biochip analyzes three of those biomarkers: MMP9, S100A6 and CST4. BICELLs based on nitrocellulose permit to immobilize antibodies for each biomarker recognition. The optical response obtained from the biosensor through the readout platform is capable to recognize specifically the desired proteins in the concentrations range for control eye (CE) and dry eye syndrome (DES). Preliminary results obtained will allow the development of a dry eye detection device useful in the area of ophthalmology and applicable to other possible diseases related to the eye dysfunction.

The Thermal and Microstructural Effect of Plasticizing HMX-Nitrocellulose Composites

DOE PAGES

Yeager, John David; Watkins, Erik Benjamin; Duque, Amanda Lynn; ...

2017-03-15

Thermal ignition via self-heating (cook-off) of cyclotetramethylene-tetranitramine (HMX)-containing plastic-bonded explosives (PBXs) is driven by the β → δ phase transition in the HMX, which is affected if not dominated by microstructure. Here, we studied the HMX-binder interface and phase transition for several variations of PBX 9404 (HMX with plasticized nitrocellulose [NC] binder). Neutron reflectometry was used to examine the interface under several conditions—pristine, after aging, and after thermal treatment. The initial interfacial structure depended on the plasticizer, but the interface homogenized over time. Thermal and optical analyses showed that all formulated materials had higher transition temperatures than neat HMX. Thismore » effect increased with NC content.« less

The Thermal and Microstructural Effect of Plasticizing HMX-Nitrocellulose Composites

NASA Astrophysics Data System (ADS)

Yeager, John D.; Watkins, Erik B.; Higginbotham Duque, Amanda L.; Majewski, Jaroslaw

2018-01-01

Thermal ignition via self-heating (cook-off) of cyclotetramethylene-tetranitramine (HMX)-containing plastic-bonded explosives (PBXs) is driven by the β → δ phase transition in the HMX, which is affected if not dominated by microstructure. Here, the HMX-binder interface and phase transition were studied for several variations of PBX 9404 (HMX with plasticized nitrocellulose [NC] binder). Neutron reflectometry was used to examine the interface under several conditions-pristine, after aging, and after thermal treatment. The initial interfacial structure depended on the plasticizer, but the interface homogenized over time. Thermal and optical analyses showed that all formulated materials had higher transition temperatures than neat HMX. This effect increased with NC content.

Air sampling methods to evaluate microbial contamination in operating theatres: results of a comparative study in an orthopaedics department.

PubMed

Napoli, C; Tafuri, S; Montenegro, L; Cassano, M; Notarnicola, A; Lattarulo, S; Montagna, M T; Moretti, B

2012-02-01

To evaluate the level of microbial contamination of air in operating theatres using active [i.e. surface air system (SAS)] and passive [i.e. index of microbial air contamination (IMA) and nitrocellulose membranes positioned near the wound] sampling systems. Sampling was performed between January 2010 and January 2011 in the operating theatre of the orthopaedics department in a university hospital in Southern Italy. During surgery, the mean bacterial loads recorded were 2232.9 colony-forming units (cfu)/m(2)/h with the IMA method, 123.2 cfu/m(3) with the SAS method and 2768.2 cfu/m(2)/h with the nitrocellulose membranes. Correlation was found between the results of the three methods. Staphylococcus aureus was detected in 12 of 60 operations (20%) with the membranes, five (8.3%) operations with the SAS method, and three operations (5%) with the IMA method. Use of nitrocellulose membranes placed near a wound is a valid method for measuring the microbial contamination of air. This method was more sensitive than the IMA method and was not subject to any calibration bias, unlike active air monitoring systems. Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Radioimmunoassays of hidden viral antigens

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

Neurath, A.R.; Strick, N.; Baker, L.

1982-07-01

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis Bmore » virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.« less

32-channel pyrometer with high dynamic range for studies of shocked nanothermites

NASA Astrophysics Data System (ADS)

Bassett, Will P.; Dlott, Dana D.

2017-01-01

A 32-channel optical pyrometer has been developed for studying temperature dynamics of shock-initiated reactive materials with one nanosecond time resolution and high dynamic range. The pyrometer consists of a prism spectrograph which directs the spectrally-resolved emission to 32 fiber optics and 32 photomultiplier tubes and digitizers. Preliminary results show shock-initiated reactions of a nanothermite composite, nano CuO/Al in nitrocellulose binder, consists of three stages. The first stage occurred at 30 ns, right after the shock unloaded, the second stage at 100 ns and the third at 1 μs, and the temperatures ranged from 2100K to 3000K. Time-resolved emission spectra suggest hot spots formed during shock unloading, which initiated the bulk thermite/nitrocellulose reaction.

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes.

PubMed

Yamaguchi, K; Asakawa, H

1988-07-01

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.

Antimicrobial activity of root canal irrigants against biofilm forming pathogens- An in vitro study

PubMed Central

Ghivari, Sheetal Basavraj; Bhattacharya, Haimanti; Bhat, Kishore G.; Pujar, Madhu A.

2017-01-01

Aims: The aim of the study was to check the antimicrobial activity of the 5% Sodium hypochlorite, 2% Chlorhexidine, 0.10% Octenidine (OCT), and 2% Silver Zeolite (SZ) at different time intervals against a single species biofilm of Enterococcus faecalis, Staphylococcus aureus, and Candida albicans model prepared on a nitrocellulose membrane. Settings and Design: In vitro nitrocellulose biofilm model was used to check antibacterial efficacy of root canal irrigants. Materials and Methods: The in vitro nitrocellulose biofilm model was used to check the antibacterial activity of root canal irrigants. Single species biofilms were suspended into 96-well microtiter plate and treated with root canal irrigants for 1, 5, 10, 15, 30, and 60 s, respectively. The remaining microbial load in the form of colony-forming unit/ml after antimicrobial treatment was tabulated and data were statistically analyzed. Statistical Analysis: SPSS version 17, Kruskal–Wallis ANOVA, Mann–Whitney U-test, and Wilcoxon matched pair test (P < 0.05) were used. Results: All tested microorganisms were eliminated within 30 s by all the antimicrobial substances tested except normal saline. 2% chlorhexidine and 0.10% OCT were equally effective against C. albicans at 30 s. Conclusion: The newly tested irrigants have shown considerable antibacterial activity against selected single species biofilm. OCT (0.10%) can be used as an alternative endodontic irrigant. PMID:29279615

Fingerprint deposition on nitrocellulose and polyvinylidene difluoride membranes using alkaline phosphatase.

PubMed

Kurien, Biji T; Danda, Debashish; Scofield, R Hal

2015-01-01

Dactyloscopy or fingerprint identification is a vital part of forensic evidence. Identification with fingerprints has been known since the finding of finger impressions on the clay surface of Babylonian legal contracts almost 4,000 years ago. The skin on the fingers and palms appears as grooves and ridges when observed under a microscope. A unique fingerprint is produced by the patterns of these friction skin ridges. Visible fingerprints can be deposited on solid surfaces. Colored inks have been used to deposit fingermarks on documents. Herein, we show that alkaline phosphatase can be used to transfer prints from fingers or palm to nitrocellulose or polyvinylidene difluoride membranes. The prints can be detected by using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate method of detection.

21 CFR 358.103 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-04-01

... (nitrocellulose) in an appropriate nonaqueous solvent that leaves a transparent cohesive film when applied to the skin in a thin layer. (c) Plaster vehicle. A fabric, plastic, or other suitable backing material in...

Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining and by tissue printing on nitrocellulose paper

PubMed Central

1987-01-01

In soybean seed coats the accumulation of the hydroxyproline-rich glycoprotein extensin is regulated in a developmental and tissue- specific manner. The time course of appearance of extensin during seed development was studied by Western blot analysis and by immunogold- silver localization. Using these techniques extensin was first detected at 16-18 d after anthesis, increasing during development to high levels at 24 d after anthesis. Immunogold-silver localization of extensin in the seed coat showed marked deposition of the glycoprotein in the walls of palisade epidermal cells and hourglass cells. The immunolocalization of extensin in developing soybean seeds was also made by a new technique--tissue printing on nitrocellulose paper. It was found that extensin is primarily localized in the seed coat, hilum, and vascular elements of the seed. PMID:3693394

Development towards Compact Nitrocellulose-Based Interferometric Biochips for Dry Eye MMP9 Label-Free In-Situ Diagnosis

PubMed Central

Santamaría, Beatriz; Laguna, María F.; López-Romero, David; Hernandez, Ana L.; Sanza, Francisco J.; Lavín, Álvaro; Casquel, Rafael; Maigler, María V.; Espinosa, Rocío L.; Holgado, Miguel

2017-01-01

A novel compact optical biochip based on a thin layer-sensing surface of nitrocellulose is used for in-situ label-free detection of metalloproteinase (MMP9) related to dry eye disease. In this article, a new integrated chip with different interferometric transducers layout with an optimal sensing surface is reported for the first time. We demonstrate that specific antibodies can be immobilized onto these transducers with a very low volume of sample and with good orientation. Many sensing transducers constitute the presented biochip in order to yield statistical data and stability in the acquired measurements. As a result, we report the recognition curve for pure recombinant MMP9, tests of model tears with MMP9, and real tear performance from patients, with a promising limit of detection. PMID:28534808

Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

Flame speed enhancement of solid nitrocellulose monopropellant coupled with graphite at microscales

NASA Astrophysics Data System (ADS)

Jain, S.; Yehia, O.; Qiao, L.

2016-03-01

The flame-speed-enhancement phenomenon of a solid monopropellant (nitrocellulose) using a highly conductive thermal base (graphite sheet) was demonstrated and studied both experimentally and theoretically. A propellant layer ranging from 20 μm to 170 μm was deposited on the top of a 20-μm thick graphite sheet. Self-propagating oscillatory combustion waves were observed, with average flame speed enhancements up to 14 times the bulk value. The ratio of the fuel-to-graphite layer thickness affects not only the average reaction front velocities but also the period and the amplitude of the combustion wave oscillations. To better understand the flame-speed enhancement and the oscillatory nature of the combustion waves, the coupled nitrocellulose-graphite system was modeled using one-dimensional energy conservation equations along with simple one-step chemistry. The period and the amplitude of the oscillatory combustion waves were predicted as a function of the ratio of the fuel-to-graphite thickness (R), the ratio of the graphite-to-fuel thermal diffusivity (α0), and the non-dimensional inverse adiabatic temperature rise (β). The predicted flame speeds and the characteristics of the oscillations agree well with the experimental data. The new concept of using a highly conductive thermal base such as carbon-based nano- and microstructures to enhance flame propagation speed or burning rate of propellants and fuels could lead to improved performance of solid and liquid rocket motors, as well as of the alternative energy conversion microelectromechanical devices.

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.

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum.

PubMed

Preechakasedkit, Pattarachaya; Pinwattana, Kulwadee; Dungchai, Wijitar; Siangproh, Weena; Chaicumpa, Wanpen; Tongtawe, Pongsri; Chailapakul, Orawon

2012-01-15

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi. Copyright © 2011 Elsevier B.V. All rights reserved.

Reflectivity Spectra for Commonly Used Reflectors

NASA Astrophysics Data System (ADS)

Janecek, Martin

2012-06-01

Monte Carlo simulations play an important role in developing and evaluating the performance of radiation detection systems. To accurately model a reflector in an optical Monte Carlo simulation, the reflector's spectral response has to be known. We have measured the reflection coefficient for many commonly used reflectors for wavelengths from 250 nm to 800 nm. The reflectors were also screened for fluorescence and angular distribution changes with wavelength. The reflectors examined in this work include several polytetrafluoroethylene (PTFE) reflectors, Spectralon, GORE diffuse reflector, titanium dioxide paint, magnesium oxide, nitrocellulose filter paper, Tyvek paper, Lumirror, Melinex, ESR films, and aluminum foil. All PTFE films exhibited decreasing reflectivity with longer wavelengths due to transmission. To achieve >;0.95 reflectivity in the 380 to 500 nm range, the PTFE films have to be at least 0.5 mm thick-nitrocellulose is a good alternative if a thin diffuse reflector is needed. Several of the reflectors have sharp declines in reflectivity below a cut-off wavelength, including TiO2 (420 nm), ESR film (395 nm), nitrocellulose (330 nm), Lumirror (325 nm), and Melinex (325 nm). PTFE-like reflectors were the only examined reflectors that had reflectivity above 0.90 for wavelengths below 300 nm. Lumirror, Melinex, and ESR film exhibited fluorescence. Lumirror and Melinex are excited by wavelengths between 320 and 420 nm and have their emission peaks located at 440 nm, while ESR film is excited by wavelengths below 400 nm and the emission peak is located at 430 nm. Lumirror and Melinex also exhibited changing angular distributions with wavelength.

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,…

Rapid diagnosis of soybean mosaic virus N soybean by tissue-print immunoassay and DIBA in comparison to other serological methods.

PubMed

Ahangaran, A; Mohammadi, Gh Mosahebi; Habibi, M Koohi; Shahraeen, N; Khezri, S

2006-01-01

Soybean mosaic virus (SMV) is an important disease in soybean and is widely distributed in northern of Iran. SMV transmitted by soybean seed and detection of it is very important for disease management. In this study, several detection methods including DAS-ELISA, indirect-ELISA, tissue-print immunoassay (TPIA) and Dot immunobinding assay (DIBA) were optimized and compared with each other to identify the virus, using polyclonal antibody. For TPIA, nitrocellulose membrane was used to imprint fresh sections of healthy and infected plant materials, and for DIBA 10 microl of extracts was doted onto nitrocellulose membranes. Both membranes were incubated 1 hour in blocking buffer, and then incubated 2 h in 1:1000 dilution of IgG-conjugate. After incubation the membranes were washed three times with PBS-T buffer for 15 min. Then the membranes were incubated in substrate solution containing NBT/BCIP. After some minutes prints or blots of infected tissues turned dark violet, whereas prints or blots of healthy ones did not show any color changes. In some cases, substrate solution was Fast red, containing 0.2M Tris-HCl buffer and 2mM MgCl2, pH = 7.8, producing red color in infected prints or blots. Both methods are simple and TPIA is rapidly and easily applicable in the field. However, TPIA had some advantages over the others. TPIA is time-saving as there is no need for conventional sap extraction and also nitrocellulose membranes used for printing can be used in the field and stored for a long time or transported to another laboratory for process. These two methods can be used routinely for detection of SMV in many samples.

Improvement of Electrical Characteristics and Stability of Amorphous Indium Gallium Zinc Oxide Thin Film Transistors Using Nitrocellulose Passivation Layer.

PubMed

Shin, Kwan Yup; Tak, Young Jun; Kim, Won-Gi; Hong, Seonghwan; Kim, Hyun Jae

2017-04-19

In this research, nitrocellulose is proposed as a new material for the passivation layers of amorphous indium gallium zinc oxide thin film transistors (a-IGZO TFTs). The a-IGZO TFTs with nitrocellulose passivation layers (NC-PVLs) demonstrate improved electrical characteristics and stability. The a-IGZO TFTs with NC-PVLs exhibit improvements in field-effect mobility (μ FE ) from 11.72 ± 1.14 to 20.68 ± 1.94 cm 2 /(V s), threshold voltage (V th ) from 1.85 ± 1.19 to 0.56 ± 0.35 V, and on/off current ratio (I on/off ) from (5.31 ± 2.19) × 10 7 to (4.79 ± 1.54) × 10 8 compared to a-IGZO TFTs without PVLs, respectively. The V th shifts of a-IGZO TFTs without PVLs, with poly(methyl methacrylate) (PMMA) PVLs, and with NC-PVLs under positive bias stress (PBS) test for 10,000 s represented 5.08, 3.94, and 2.35 V, respectively. These improvements were induced by nitrogen diffusion from NC-PVLs to a-IGZO TFTs. The lone-pair electrons of diffused nitrogen attract weakly bonded oxygen serving as defect sites in a-IGZO TFTs. Consequently, the electrical characteristics are improved by an increase of carrier concentration in a-IGZO TFTs, and a decrease of defects in the back channel layer. Also, NC-PVLs have an excellent property as a barrier against ambient gases. Therefore, the NC-PVL is a promising passivation layer for next-generation display devices that simultaneously can improve electrical characteristics and stability against ambient gases.

Paper membrane-based SERS platform for the determination of glucose in blood samples.

PubMed

Torul, Hilal; Çiftçi, Hakan; Çetin, Demet; Suludere, Zekiye; Boyacı, Ismail Hakkı; Tamer, Uğur

2015-11-01

In this report, we present a paper membrane-based surface-enhanced Raman scattering (SERS) platform for the determination of blood glucose level using a nitrocellulose membrane as substrate paper, and the microfluidic channel was simply constructed by wax-printing method. The rod-shaped gold nanorod particles were modified with 4-mercaptophenylboronic acid (4-MBA) and 1-decanethiol (1-DT) molecules and used as embedded SERS probe for paper-based microfluidics. The SERS measurement area was simply constructed by dropping gold nanoparticles on nitrocellulose membrane, and the blood sample was dropped on the membrane hydrophilic channel. While the blood cells and proteins were held on nitrocellulose membrane, glucose molecules were moved through the channel toward the SERS measurement area. Scanning electron microscopy (SEM) was used to confirm the effective separation of blood matrix, and total analysis is completed in 5 min. In SERS measurements, the intensity of the band at 1070 cm(-1) which is attributed to B-OH vibration decreased depending on the rise in glucose concentration in the blood sample. The glucose concentration was found to be 5.43 ± 0.51 mM in the reference blood sample by using a calibration equation, and the certified value for glucose was 6.17 ± 0.11 mM. The recovery of the glucose in the reference blood sample was about 88 %. According to these results, the developed paper-based microfluidic SERS platform has been found to be suitable for use for the detection of glucose in blood samples without any pretreatment procedure. We believe that paper-based microfluidic systems may provide a wide field of usage for paper-based applications.

Hazardous Waste Cleanup: Hercules Incorporated in Parlin, New Jersey

EPA Pesticide Factsheets

Hercules, Inc. is located at 50 South Minisink Avenue in Parlin, New Jersey. The plant started operations in the early 1900's at a 670-acre site adjacent to the Sayreville watershed. Its main product through the years has been nitrocellulose, which was use

21 CFR 73.2329 - Guanine.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose. (b...

21 CFR 73.2329 - Guanine.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose. (b...

21 CFR 73.2329 - Guanine.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose. (b...

Electrospin-coating of nitrocellulose membrane enhances sensitivity in nucleic acid-based lateral flow assay.

PubMed

Yew, Chee-Hong Takahiro; Azari, Pedram; Choi, Jane Ru; Li, Fei; Pingguan-Murphy, Belinda

2018-06-07

Point-of-care biosensors are important tools developed to aid medical diagnosis and testing, food safety and environmental monitoring. Paper-based biosensors, especially nucleic acid-based lateral flow assays (LFA), are affordable, simple to produce and easy to use in remote settings. However, the sensitivity of such assays to infectious diseases has always been a restrictive challenge. Here, we have successfully electrospun polycaprolactone (PCL) on nitrocellulose (NC) membrane to form a hydrophobic coating to reduce the flow rate and increase the interaction rate between the targets and gold nanoparticles-detecting probes conjugates, resulting in the binding of more complexes to the capture probes. With this approach, the sensitivity of the PCL electrospin-coated test strip has been increased by approximately ten-fold as compared to the unmodified test strip. As a proof of concept, this approach holds great potential for sensitive detection of targets at point-of-care testing. Copyright © 2018 Elsevier B.V. All rights reserved.

Lipid-binding analysis using a fat blot assay.

PubMed

Munnik, Teun; Wierzchowiecka, Magdalena

2013-01-01

Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose membrane-immobilized lipids. The assay is adapted from the method by Dowler et al. (Sci STKE 129:pl6, 2002) and provides qualitative and quantitative information on the relative affinity with which a protein binds to a particular lipid. To perform a Fat Blot assay, serial dilutions of different phospholipids are spotted onto a nitrocellulose membrane. These membranes are then incubated with a lipid-binding protein possessing a GST (or other epitope) tag. The membranes are washed and the protein, which is bound to the membrane by virtue of its interaction with the lipid's head group, is detected by immunoblotting with an antibody against GST (or other epitope). The procedure only requires a few micrograms of protein and is quick, simple and cheap to perform.

Mid-infrared matrix assisted laser desorption ionization with a water/glycerol matrix

NASA Astrophysics Data System (ADS)

Caldwell, Kathleen L.; Murray, Kermit K.

1998-05-01

Matrix-assisted laser desorption ionization (MALDI) mass spectra were obtained using a water and glycerol matrix with a tunable mid-infrared optical parametric oscillator. The matrix consists of a 1:1 mixture of water and glycerol deposited on a thin layer of nitrocellulose and cooled to -30°C. When exposed to vacuum, most of the water evaporates, leaving a matrix of glycerol with residual water. The peptide bradykinin and the protein bovine insulin were used to test this new matrix. Mass spectra were obtained for bradykinin between 2.76 and 3.1 μm with the maximum analyte signal at 2.8 μm. Mass resolution in excess of 2000 for bradykinin and 500 for insulin was obtained with delayed ion extraction and a linear time of flight mass spectrometer. The addition of nitrocellulose to the matrix resulted in exceptionally durable samples: more than 10,000 laser shots which produced analyte signal could be obtained from a single sample spot.

Western blotting using chemiluminescent substrates.

PubMed

Alegria-Schaffer, Alice

2014-01-01

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.

Regenerative Medicine and Restoration of Joint Function

DTIC Science & Technology

2013-10-01

examined if the inability of adult human chondrocytes to form cartilage tissue is dependent on age or species differences between human and cows ...SDS-PAGE, and transferred to a nitrocellulose membrane by iblot (Invitrogen, US). Membranes were blocked in 1% milk for 1 h and incubated with

Fused slurry silicide coatings for columbium alloy reentry heat shields. Volume 2: Experimental and coating process details

NASA Technical Reports Server (NTRS)

Fitzgerald, B.

1973-01-01

The experimental and coating process details are presented. The process specifications which were developed for the formulation and application of the R-512E fused slurry silicide coating using either an acrylic or nitrocellulose base slurry system is also discussed.

16 CFR 1611.31 - Terms defined.

Code of Federal Regulations, 2014 CFR

2014-01-01

... FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.31 Terms defined. As used in this part, unless... material subject to the act, except film and fabrics having a nitro-cellulose fiber, finish, or coating... pile, nap, or tufting. (i) The term film means any nonrigid, unsupported plastic, rubber or other...

16 CFR 1611.31 - Terms defined.

Code of Federal Regulations, 2012 CFR

2012-01-01

... FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.31 Terms defined. As used in this part, unless... material subject to the act, except film and fabrics having a nitro-cellulose fiber, finish, or coating... pile, nap, or tufting. (i) The term film means any nonrigid, unsupported plastic, rubber or other...

16 CFR 1611.31 - Terms defined.

Code of Federal Regulations, 2010 CFR

2010-01-01

... FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.31 Terms defined. As used in this part, unless... material subject to the act, except film and fabrics having a nitro-cellulose fiber, finish, or coating... pile, nap, or tufting. (i) The term film means any nonrigid, unsupported plastic, rubber or other...

Alternate Methods for Disposal of Nitrocellulose Fines

DTIC Science & Technology

1985-07-22

13 Microwave ..................................... 14 Plasma ........................................ 14V Laser pyrolysis...would either be backflushed (not expected to be too successful) or replaced. Microwave Thermal Degradation The use of microwave heating has been...with microwave heating, new designs would be needed if a practical, cost effective system is to be developed. Considerable additional research would be

16 CFR 1610.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Test procedures for textile fabrics and film... for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose fiber... under the procedures outlined in part 1611, Standard for the Flammability of Vinyl Plastic Film, and if...

16 CFR 1610.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Test procedures for textile fabrics and film... for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose fiber... under the procedures outlined in part 1611, Standard for the Flammability of Vinyl Plastic Film, and if...

16 CFR § 1611.31 - Terms defined.

Code of Federal Regulations, 2013 CFR

2013-01-01

... FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.31 Terms defined. As used in this part, unless... material subject to the act, except film and fabrics having a nitro-cellulose fiber, finish, or coating... pile, nap, or tufting. (i) The term film means any nonrigid, unsupported plastic, rubber or other...

16 CFR 1611.37 - Reasonable and representative tests under section 8 of the Act.

Code of Federal Regulations, 2014 CFR

2014-01-01

... FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations..., on initial test a film or a textile fabric with a nitro-cellulose fiber, finish or coating, does not... production shall be deemed reasonable and representative tests for such film or textile fabric. (d...

16 CFR 1610.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Test procedures for textile fabrics and film... for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose fiber... under the procedures outlined in part 1611, Standard for the Flammability of Vinyl Plastic Film, and if...

16 CFR 1611.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 16 Commercial Practices 2 2012-01-01 2012-01-01 false Test procedures for textile fabrics and film... REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.33 Test procedures for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose...

16 CFR 1611.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 2 2011-01-01 2011-01-01 false Test procedures for textile fabrics and film... REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.33 Test procedures for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose...

16 CFR 1611.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 16 Commercial Practices 2 2014-01-01 2014-01-01 false Test procedures for textile fabrics and film... REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.33 Test procedures for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose...

16 CFR 1610.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Test procedures for textile fabrics and film... for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose fiber... under the procedures outlined in part 1611, Standard for the Flammability of Vinyl Plastic Film, and if...

16 CFR 1611.33 - Test procedures for textile fabrics and film.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Test procedures for textile fabrics and film... REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations § 1611.33 Test procedures for textile fabrics and film. (a)(1) All textile fabrics (except those with a nitro-cellulose...

16 CFR 1611.37 - Reasonable and representative tests under section 8 of the Act.

Code of Federal Regulations, 2010 CFR

2010-01-01

... FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and Regulations..., on initial test a film or a textile fabric with a nitro-cellulose fiber, finish or coating, does not... production shall be deemed reasonable and representative tests for such film or textile fabric. (d...

A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

ERIC Educational Resources Information Center

Chang, Ming-Mei; Lovett, Janice

2011-01-01

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

Measuring the Speed of Sound through Gases Using Nitrocellulose

ERIC Educational Resources Information Center

Molek, Karen Sinclair; Reyes, Karl A.; Burnette, Brandon A.; Stepherson, Jacob R.

2015-01-01

Measuring the heat capacity ratios, ?, of gases either through adiabatic expansion or sound velocity is a well established physical chemistry experiment. The most accurate experiments depend on an exact determination of sound origin, which necessitates the use of lasers or a wave generator, where time zero is based on an electrical trigger. Other…

Saturation Transfer Difference NMR as an Analytical Tool for Detection and Differentiation of Plastic Explosives on the Basis of Minor Plasticizer Composition

DTIC Science & Technology

2015-05-01

HMX ); ethylene glycol dinitrate (EGDN); ammonium nitrate (AN); and nitrocellulose (NC).1–4 Alternatively, in one recent study,5 fluorescence-based...saturation transfer difference AN ammonium nitrate BSA bovine serum albumin EGDN ethylene glycol dinitrate HDO partially deuterated water HMX

Synthetic microfluidic paper: high surface area and high porosity polymer micropillar arrays.

PubMed

Hansson, Jonas; Yasuga, Hiroki; Haraldsson, Tommy; van der Wijngaart, Wouter

2016-01-21

We introduce Synthetic Microfluidic Paper, a novel porous material for microfluidic applications that consists of an OSTE polymer that is photostructured in a well-controlled geometry of slanted and interlocked micropillars. We demonstrate the distinct benefits of Synthetic Microfluidic Paper over other porous microfluidic materials, such as nitrocellulose, traditional paper and straight micropillar arrays: in contrast to straight micropillar arrays, the geometry of Synthetic Microfluidic Paper was miniaturized without suffering capillary collapse during manufacturing and fluidic operation, resulting in a six-fold increased internal surface area and a three-fold increased porous fraction. Compared to commercial nitrocellulose materials for capillary assays, Synthetic Microfluidic Paper shows a wider range of capillary pumping speed and four times lower device-to-device variation. Compared to the surfaces of the other porous microfluidic materials that are modified by adsorption, Synthetic Microfluidic Paper contains free thiol groups and has been shown to be suitable for covalent surface chemistry, demonstrated here for increasing the material hydrophilicity. These results illustrate the potential of Synthetic Microfluidic Paper as a porous microfluidic material with improved performance characteristics, especially for bioassay applications such as diagnostic tests.

Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

PubMed

de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

2017-01-18

We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, K D eff . Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

PubMed Central

Paton, B C; Poulos, A

1988-01-01

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands. Images Fig. 1. Fig. 2. Fig. 3. PMID:3178760

[Receptor elements for biosensors in two ways of methylotrophic yeast immobilization].

PubMed

Zaĭtsev, M G; Arliapov, V A; Alferov, V A; Reshetilov, A N

2012-01-01

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 In yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05-1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2-1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzochinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.

16 CFR § 1611.37 - Reasonable and representative tests under section 8 of the Act.

Code of Federal Regulations, 2013 CFR

2013-01-01

... COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE FLAMMABILITY OF VINYL PLASTIC FILM Rules and..., on initial test a film or a textile fabric with a nitro-cellulose fiber, finish or coating, does not... production shall be deemed reasonable and representative tests for such film or textile fabric. (d...

Field Demonstration Report Applied Innovative Technologies for Characterization of Nitrocellulose- and Nitroglycerine Contaminated Buildings and Soils, Rev 1

DTIC Science & Technology

2007-01-05

positive / false negatives. The quantitative on-site methods were evaluated using linear regression analysis and relative percent difference (RPD) comparison...Conclusion ...............................................................................................3-9 3.2 Quantitative Analysis Using CRREL...3-37 3.3 Quantitative Analysis for NG by GC/TID.........................................................3-38 3.3.1 Introduction

Passage of Campylobacter jejuni and Campylobacter coli subtypes through 0.45 and 0.65 µm pore size nitro-cellulose filters

USDA-ARS?s Scientific Manuscript database

Campylobacter can be difficult to recover from complex samples due to overgrowth by background bacteria. A 0.45 or 0.65 µm pore size filter overlaid on agar plates can be used as a means to separate Campylobacter from confounding non-Campylobacter cells, facilitating detection on solid plating medi...

Method of producing thin cellulose nitrate film

DOEpatents

Lupica, S.B.

1975-12-23

An improved method for forming a thin nitrocellulose film of reproducible thickness is described. The film is a cellulose nitrate film, 10 to 20 microns in thickness, cast from a solution of cellulose nitrate in tetrahydrofuran, said solution containing from 7 to 15 percent, by weight, of dioctyl phthalate, said cellulose nitrate having a nitrogen content of from 10 to 13 percent.

Laboratory Tests to Determine the Chemical and Physical Characteristics of Propellant-Solvent-Fuel Oil Mixtures

DTIC Science & Technology

1990-02-01

Decomposition ................ 165 Part IV. Thermal Decomposition - Analytical Methodologies .............. 167 Part V. Miscellaneous...500C ................... 45 12 Differential Scanning Calorimetry Curve for the Decomposition of a Smokeless-Grade Nitrocellulose .......... 62 13 Process...cellulose backbone with nitrating acids of high water content resulted in hydrolysis of the pentosans without the desired 3 result of nitration. Furthermore

Environmental Acceptable Medium Caliber Ammunition Percussion Primers

DTIC Science & Technology

2008-05-01

the nanoparticles extremely hydrophobic. The alternative treatment of the solution was the addition of ammonium dihydrogen phosphate (ADP) to serve as...Ultrafine Aluminum Nanoparticles," LA-UR-04-2921. 49 ACRONYM LIST ADP Ammonium Dihydrogen Phosphate Al Aluminum ARDEC Armament Research Development and...Nitrocellulose Nd:Yag Neodymium -doped yttrium aluminum garnet NSWC-IH Naval Surface Warfare Center- Indian Head PAD Propellant actuated device PETN

Intelligent Computation for Optimal Fabrication Condition of a Protein Chip with Ni-Co Alloy-Coated Surface.

PubMed

Chang, Yaw-Jen; Chang, Cheng-Hao

2016-06-01

Based on the principle of immobilized metal affinity chromatography (IMAC), it has been found that a Ni-Co alloy-coated protein chip is able to immobilize functional proteins with a His-tag attached. In this study, an intelligent computational approach was developed to promote the performance and repeatability of a Ni-Co alloy-coated protein chip. This approach was launched out of L18 experiments. Based on the experimental data, the fabrication process model of a Ni-Co protein chip was established by using an artificial neural network, and then an optimal fabrication condition was obtained using the Taguchi genetic algorithm. The result was validated experimentally and compared with a nitrocellulose chip. Consequentially, experimental outcomes revealed that the Ni-Co alloy-coated chip, fabricated using the proposed approach, had the best performance and repeatability compared with the Ni-Co chips of an L18 orthogonal array design and the nitrocellulose chip. Moreover, the low fluorescent background of the chip surface gives a more precise fluorescent detection. Based on a small quantity of experiments, this proposed intelligent computation approach can significantly reduce the experimental cost and improve the product's quality. © 2015 Society for Laboratory Automation and Screening.

Colorimetric stack pad immunoassay for bacterial identification.

PubMed

Eltzov, Evgeni; Marks, Robert S

2017-01-15

A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (10 2 cellsmL -1 ). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

Analytical Method for Determining Tetrazene in Water.

DTIC Science & Technology

1987-12-01

8217-decanesulfonic acid sodium salt. The mobile phase pH was adjusted to 3 with glacial acetic acid. The modified mobile phase was optimal for separating of...modified with sodium tartrate, gave a well-defined reduction wave at the dropping mercury electrode. The height of the reduction wave was proportional to...anitmony trisulphide, nitrocellulose, PETN, powdered aluminum and calcium silicide . The primer samples were sequentially extracted, first with

Elution of viruses by ionic and nonionic surfactants.

PubMed Central

Fujito, B T; Lytle, C D

1996-01-01

The ionic and nonionic surfactants sodium dodecyl sulfate and Triton X-100, respectively, eluted two viruses, phi X174 and PRD1, which were adsorbed to the ionic and nonionic binding membranes cationic polysulfone and nitrocellulose, respectively. Results indicated that complete elution was readily achieved only when combinations of surfactants and binding membranes were matched (i.e., ionic-ionic or nonionic-nonionic). PMID:8795240

Regenerative Stem Cell Therapy for Breast Cancer Bone Metastasis

DTIC Science & Technology

2015-11-01

nitrocellulose membranes (Millipore) followed by blocking with 2% non- fat milk and incubation with primary antibodies, overnight at 4C. The b-actin...TNF-like proteins : Osteoprotegerin (OPG), RANK and RANKL, which together regulate osteoclast function (1). The dysregulation of the functional...among proteins in the same family are an indication they may have functional importance, so these residues may be important in mediating the

Environmental Quality Standards Research on Wastewaters of Army Ammunition Plants

DTIC Science & Technology

1978-06-01

characterization of nitrocellulose wastewaters. We are grateful to LTC Leroy H. Reuter and LTC Robert P. Carnahan of the US Army Medical Research and...analytical methodology was required to characterize the wastes. The techniques used for fingerprinting (showing that the same compound exists although its...examination of the NC wastewaters has somewhat clarified the problem of characterizing the NC and has caused us to change or modify previous

Blotting from PhastGel to Membranes by Ultrasound.

PubMed

Kost, Joseph; Azagury, Aharon

2015-01-01

Ultrasound based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Blotting from PhastGel to membranes by ultrasound.

PubMed

Kost, Joseph

2009-01-01

Ultrasound-based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and (14)C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Diagnosis of AIDS-Related Intestinal Parasites

DTIC Science & Technology

1988-01-07

malnutrition or with certain concomitant illnesses such as measles or chicken pox , may be susceptible to more severe clinical manifestations with cure only...above) and Homero Martinez, M.D., Instituto National de la Nutricion, Mexico City. In this study, 53 children with at least one microscopic stool...outlined or using nitrocellulose "DOT-blot" technology. In addition, plans are underway to begin field testing the Entamoeba histolytica ELISA in Mexico

Conference Proceedings on Smokeless Propellants Held in Florence, Italy on 12-13 September 1985

DTIC Science & Technology

1986-01-01

i) propellants for tactical missiles, either the introduction of sufficient amounts of such nitrocellulose or an equivalent alternative ingredient to...applications have been developed. Nr departure from conventional EDB processing is involved. 1. INTRODUCTION 1.1 Nitramines, RDX and HMX, are dense, energetic...cycling. The introduction of nitramine is in most cases accompanied by changes in nitrocel- lulose/plasticiser ratio. Thus the physical properties

Bomb Detection System Study

DTIC Science & Technology

1965-01-01

cause the reduction in vapor pressure. To determine whether an explosive can be detected thi ugh its * vapor, it is necessary to know the vapor pressures...Smokeless powders are explosives normally used in shotgun powders and can vary widely in composition. Nitrocellulose is the major :oomponent. Dinitrotoluene...will not be inclined to undertake the risky process except under very unusual circum- stances. Black powder is an obvious choice and has been so in

Propellant Reuse/Recovery Technology

DTIC Science & Technology

1988-08-31

viscosity of the nitrocellulose (NC) determine the solvent/solvent and solvent/propellant ratios required to properly resolvate the propellant. It was also...plasticization. An 11-min drying cycle was required to remove the excess solvent from the over-solvated propellant. To improve plasticization using...solvent, and (4) 15-min mix cycle. To eliminate the drying cycle and determine that a 15-min mix cycle will resolvate the propellart, an additional 1 h

Combustible Cartridge Case Characterization

DTIC Science & Technology

1984-02-01

Latex 241 * Nitrocellulose and Kraft /fibers National Starch Resin 78-3730 The beater additive process for manufacturing combustible cartridge cases *is... Kraft Fibers The Kraft fibers were received as sheets weighing approximately 1.3 lb each. The moisture content of the sheets varied from 6 to 7-1/2... Kraft fibers was incorporated into each batch. Resins The resins were supplied as a water emulsion with nominally 50 percent solids. Samples of each

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.

Effects of Near Field Pyroshock on the Performance of a Nitramine Nitrocellulose Propellant

NASA Technical Reports Server (NTRS)

Baca, Arcenio B.

2016-01-01

The overall purpose of this study is to investigate the effects of a pyroshock environment on the performance characteristics of a propellant used in pyrotechnic devices such as guillotine cutters. Near field pyroshock which is defined by acceleration amplitudes in excess of 10,000g at a frequency of greater than 10,000 Hz is a highly transient environment that has a known potential to cause failure in both structural and electronic components. A heritage pressure cartridge assembly which uses a nitramine nitrocellulose propellant with a known performance baseline will be exposed to a near field pyroshock event. The pressure cartridge will then be fired in an ambient closed bomb firing to collect pressure time history. The two performance characteristics that will be evaluated are the pressure amplitude and time to peak pressure. This data will be compared to the base-lined ambient closed bomb data to evaluate the effects of the shock on the performance of the propellant. It is expected that the pyroshock environment will cause brittle failures of the propellant increasing the surface area of said propellant. This increase of surface area should result in increased combustion rate which should show as an increased pressure peak and decreased time to peak pressure in the pressure time data.

Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes.

PubMed

Ruan, Guang-Ping; Ma, Li; Meng, Xiao-Jing; Meng, Min-Jie; Wang, Xiao-Ning; Lin, Ying; Wu, Zheng-Qiang; He, Xiao-Wei; Wang, Ju-Fang; Zhu, Yong

2007-01-01

HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.

Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots.

PubMed

Domingo, A; Marco, R

1989-10-01

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.

Investigating The Anti-apoptotic Effects of Shigella Flexneri Infection In Epithelial Cells

DTIC Science & Technology

2009-08-13

also been found in breast milk of convalescent mothers and most likely contribute to the reduction of disease severity in breast-fed infants (45...samples. Proteins were transferred to a nitrocellulose membrane and blocked with 5% dry milk in Tris-buffered saline (TBS). Caspases were detected by...dry milk overnight at 4°C. After washing, donkey anti- rabbit immunoglobulin G antibody conjugated to horseradish peroxidase (Amersham Biosciences

Role of Reactive Stroma in Prostate Cancer Progression

DTIC Science & Technology

2008-02-01

gel. Proteins were transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) and incubated in PBS buffer with 5% nonfat milk at...fusion protein, and incubated for 2 hours at room temperature. Secondary antibody was biotin-conjugated sheep anti-mouse IgG (Sigma), diluted at 1...stroma that forms at sites of wound repair, microbial invasion, or carcinoma as we have reported previously (1, 32, 35). Of these, CTGF is a known

Targeting Estrogen Receptor-Beta in Triple-Negative Breast Cancer

DTIC Science & Technology

2014-04-01

polyacrylamide gels. Proteins were transferred to a nitrocellulose membrane for 1.5 hr at 0.35 A. Membranes were blocked with 5% nonfat milk and...PerkinElmer, Waltham, MA). Total protein was measured using the Bradford Method (Bio-Rad), and raw luciferase data were normalized to total protein ...are expressed as fold induction of raw luciferase units per mg protein over the DMSO control ± SD. Experiments were repeated at least twice. *p

An Exploratory Investigation of the Influence of Igniter Chemistry on Ignition in Porous Bed Gun Propellants

DTIC Science & Technology

1981-09-01

nitrocellulose igniter materials using the Ignition Energetics Characterization Device (IECD). The results presented herein represent Phase II eperimental ...auxiliary test cham- bcz is a combustion gas diagnostic section designed to permit determination of the composition and enthalpy level of the gases...removal/assembly and propellant loading. 2.2 Igniter Characteristics 2.2.1 Baseline Igniter The igniter system, Figure 2.3, is designed to provide overall

Breast Cancer and Estrogen Biosynthesis in Adipose Tissue

DTIC Science & Technology

1998-10-01

transferred to a nitrocellulose mem - brane. The transferred proteins were subjected to a denaturation/rena- turation process and hybridized to the 32P...aromatase expression in adipose tissue has been recently observed to be regulated by mem - bers of the interleukin-6 (IL-6) cytokine family. Based on...shown in human adipose stromal cells that the stimulatory effects of serum on aromatase expression can be mimicked by mem - bers of the interleukin-6

Biomedical Applications of Micro-Raman and Surface-Enhanced Raman Scattering (SERS) Technology

DTIC Science & Technology

2012-10-01

to be an effective media for PSA capture. For SERS-based immunoassays, nitrocellulose offers comparable results to those obtained using gold-coated...glass substrates while offering a more cost- effective and time-saving method of detecting minute amounts of PSA; (ii) Micro-Raman imaging...technology was found to be effective in chemical mapping of arteries in the tissues of a post mortem individual whose cause of death was a cardiac event

Dissolution Rate of Propellant Energetics from Nitrocellulose Matrices

DTIC Science & Technology

2012-09-01

propellants are generally composed of a polymer, a plasticizer , and a stabilizer. These three compo- nents provide the structure, contribute the oxygen...million lb) of nitrocel- lulose is manufactured each year in the U.S. (Richie 2012; a partial listing is given in Table A1). This wood -like compound...that single- and double-base propellants are transparent to translucent and show signs of having been extruded (Fig. 10). Figure 10. Thin section

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.

Continuous Manufacturing of Nitrocellulose by Magnesium Nitrate Method. Volume 1

DTIC Science & Technology

1979-06-01

enters a scrubber . The scrubber removes entrained acid, water, and NC fines from the air before it enters a Roots water sealed (lobe type) vacuum pump...and is exhausted to the atmosphere. The air enters the bottom of the scrubber and is forced (by vacuum) sequentially through two weir arrangements...the panel from left to right, the Eimco dewatering filter drive, vacuum pressure, receiver, vacuum scrubber , and pump controls may be seen along with

Liquid Chromatographic Analysis of Nitrocellulose-Base Propellants

DTIC Science & Technology

1983-03-28

18 radial PkC column using 10 micron silica coated with octadecyl silane. The detector was a LC 85 variable wavelength UV detector from Perkin-Elmer...Snyder, L. R. J Chrom Scd i6, (1978), 223. i •2 Glatch, J. L., Kirkland, J. J., Squire, K. M., and Miner, J. M.; J. Chromatgography, 199, (1980), 57. 4...assuming non retained peaks elute at 1 minute). This produces 3 solvents of approximately the same strengths bnt greatly differing selectivities. Once

Analysis and Characterization of High-Purity Talc for Use in Propellants; Determination of Talc in Propellants

DTIC Science & Technology

1975-12-01

is determined by combustion in an induction furnace with iron as a flux. The methods for moisture, loss in ignition, water-soluble matter, acid... determination of talc in nitro- cellulose-base propellants. The first method (which is the method recom- mended for the usual nitrocellulose -base...In the present report an improved scheme is proposed for the analysis of talc. The silica and magnesium oxide are determined by fusion with sodium

Human Progesterone A-Form as a Target for new Drug Discovery in Human Breast Cancer

DTIC Science & Technology

1999-07-01

Development of Dissociated Antiprogestins. Endocrinol., 140: 1449-1458. Giangrande, P.H., and McDonnell, D.P. (1999). The A and B isoforms of the human...from Schering Pharmaceuticals (Berlin, Germany). Secondary antibodies, Hybond-C Extra (nitrocellulose) transfer membrane, and developing film were...by J. D. Chen (University of Massachusetts, Worcester, MA); pGEX-5X-1 was obtained from Pharmacia Biotech (Uppsala, Sweden); pGEX.1-GRIP 1 was provided

Investigation of the Effectiveness of Polymers in the Treatment of Nitrocellulose-Manufacturing Wastewater

DTIC Science & Technology

1976-03-01

Nonionic polyacryl - aaide was the most effective for the purification and up to 80% removal of pulp fiber was obtained In a wide range of pH tested...Anionic polyolectrolyte. such as 3odium alginate and sodium polyacrylate , however, dispersed the suspension of pulp fiber instead of coagulating it. The...This process is called filter conditioning. They have observed that the continuous application of a polyacryl - amide solution (5-50 ppb) improved the

Degradation of TATP, TNT, and RDX using mechanically alloyed metals

NASA Technical Reports Server (NTRS)

Geiger, Cherie (Inventor); Sigman, Michael (Inventor); Fidler, Rebecca (Inventor); Clausen, Christian (Inventor)

2012-01-01

Bimetallic alloys prepared in a ball milling process, such as iron nickel (FeNi), iron palladium (FePd), and magnesium palladium (MgPd) provide in situ catalyst system for remediating and degrading nitro explosive compounds. Specifically, munitions, such as, 2,4,6-trinitrotoluene (TNT), cyclo-1,3,5-trimethylene-2,4,6-trinitramine (RDX), nitrocellulose and nitroglycerine that have become contaminants in groundwater, soil, and other structures are treated on site to remediate explosive contamination.

Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes.

PubMed

Kurien, Biji T; Scofield, R Hal

2002-08-01

Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electro-transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins.

Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis.

PubMed Central

Zone, J J; Taylor, T B; Kadunce, D P; Meyer, L J

1990-01-01

Linear IgA bullous dermatosis (LABD) is a rare blistering skin disease characterized by basement membrane zone deposition of IgA. This study identifies a tissue antigen detected by patient serum and then isolates the autoantibody using epidermis and protein bands blotted on nitrocellulose as immunoabsorbents. Sera from 10 patients (9 with cutaneous disease and 1 with cicatrizing conjunctivitis) were evaluated. Indirect immunofluorescence revealed an IgA anti-basement membrane antibody in 6 of 10 sera with monkey esophagus substrate and 9 of 10 sera with human epidermal substrate. Immunoblotting was performed on epidermal and dermal extracts prepared from skin separated at the basement membrane zone with either sodium chloride or EDTA. Saline-separated skin expressed a 97-kD band in dermal extract alone that was recognized by 4 of 10 sera. EDTA-separated skin expressed the 97-kD band in both epidermal (4 of 10 sera) and dermal (6 of 10 sera) extract. Immunoabsorption of positive sera with epidermis purified an IgA antibody that reacted uniquely with the 97-kD band. In addition, IgA antibody bound to nitrocellulose was eluted from the 97-kD band and found to uniquely bind basement membrane zone. It is likely that the 97-kD protein identified by these techniques is responsible for basement membrane binding of IgA in LABD. Images PMID:2107211

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes.

PubMed

Lin, J C; Pagano, J S

1986-08-01

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.

Identification of miRNA Signatures Associated with Epithelial Ovarian Cancer Chemoresistance with Further Biological and Functional Validation of Identified Key miRNAs

DTIC Science & Technology

2012-08-01

separated on 12% SDS PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% non- fat milk (Labscientific, Inc) in TBS-Tween buffer... Raw mass spectrometric data were processed and analyzed for variations in the spectral counts of peptides between sample sets and bioinformatics was...accomplished using Ingenuity Pathways Analysis (IPA). Results: The total numbers of proteins and peptides identified are listed in the table

Water Quality Criteria for Nitrocellulose

DTIC Science & Technology

1986-06-01

38 . 6. REFERENCES. .................................................... 40 ’ 7. GLOSSARY...Flexible "collodion, FM--NIS, Guncotton, IX 3/5, Kodak Li 115, LR 115, Nitrocel- lulose E950, Nitrocotton, Nitron , Nixon N/C, NTs 62, Nrs 218, Nrs 222...0OCf 38.79 15.8 40 ,827h 24 mo 1 42.69 6.5 1,526 24 mo 3 NAI 6.7 NDJ 24 mo 10 38.35 13.8 36,031k Mouse 24 mo 0 37 6.2 0 (female) 24 ma 1of 34.2 12.8

Synthesis and Characterization of Energetic Plasticizer AMDNNM

NASA Astrophysics Data System (ADS)

Schulze, Maxwell C.; Chavez, David E.

2016-04-01

The synthesis of room temperature liquid azidomethyl-dinitroxydimethyl-nitromethane (AMDNNM, 5) in 57% overall yield and its formulation with nitrocellulose (AMDNNM/NC) are described. The small-scale explosive sensitivity of neat AMDNNM was determined to be slightly more sensitive than PETN, whereas AMDNNM/NC is significantly less sensitive. Both neat AMDNNM and AMDNNM/NC have thermal stabilities similar to that of pentaerythritol tetranitrate (PETN). The explosive and chemical properties of this novel material make it a good candidate for an energetic plasticizer.

Decontamination of Explosives-Contaminated Range Scrap Using a Transportable Hot Gas Decontamination System

DTIC Science & Technology

2003-05-01

I I I I FINAL • Composition B (TNT, RDX and wax), • Tetryl, • Smokeless Powder ( Nitrocellulose /Nitrogylcerin), and • HBX (TNT, RDX, aluminum...operating-cost system. Because of its temporary, on-site configuration, this is an inherently low-cost method to decontaminate range residue. On... methods ) were evaluated and progressively improved during each test run. 022/masterdocument.doc 4-12 I I I I I I I I I I I I I I I I

Basic Rheology and Its Application to Nitrocellulose Propellant Processing by Screw Mix-Extruders

DTIC Science & Technology

1990-09-01

plastics and rubber industries. In its raw state NC retains much of the supermolecular structure of the precursor cellulose , and it exists in the form of...they have a cholesteric liquid crystal structure, in common with many other cellulosic materials(ref.8). It has been postulated that thermal...and molecules, see figure 19. Fibrils are about 25 plm in diameter, and are made up of ordered bundles of microfibrils which are about 3 jim in

Applied Innovative Technologies for Characterization of Nitrocellulose and Nitroglycerin Contaminated Buildings and Soils

DTIC Science & Technology

2008-07-01

analyses of the NG test group samples are summarized in Table 4- 8 along with results for the lab reference method, STL (SW- 846 ) Method 8330. The results for...Drive, Suite 17D08,Alexandria,VA,22350-3605 8 . PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10...Standard Form 298 (Rev. 8 -98) Prescribed by ANSI Std Z39-18 i COST & PERFORMANCE REPORT Project: ER-0130 TABLE OF CONTENTS Page 1.0 EXECUTIVE

Detailed Characterization of Hypervelocity Firings in a Long 120-MM Gun

DTIC Science & Technology

1991-03-01

Figure 14. Experinental Pressure-Time Curves for Round2L Qf S d Phase Firing Seris.± 14 series led to similarly good agreement in modeling the second...Applied Ballistics Branch, and Messrs. R. May, D . Meier, and S. Little of Applied Concets Corporation are acknowledged for their uEual high level of...LB IfG AT aCTRACT RAOFORD ARMY AMUNITION PLANT. RADFORO, VA. D rWAOM9- .-Z-0003 NITROCELLULOSE - ACCEPTED BLEND NUMB I NITROGEN CNTENT 1 KI STARCH I

Insulation of Nitrocellulose Boiling Tubs at Radford Army Ammunition Plant

DTIC Science & Technology

1982-03-01

control system. The amount of steam usea for the on-boil cycle with the single-sensor autocontrol averaged 647 kg/hr (1426 lb/hr) (test 1, table 2...This was a reduc- tion of 210 kg/hr (463 lb/hr) over the manually controlled uninsulated tub. Steam usage with the single sensor autocontrol and...uninsulated tub. At times durin)g the on- boil cycle of tests I and 2, the temperature of the manual sensor was different from the autocontrol sensor indicating

The Molecular Anatomy of PFDA Hepatotoxicity as Studied by Two-Dimensional Electrophoresis

DTIC Science & Technology

1993-02-26

indicators of cell injury and dysfunction. The compound chosen initially, perfluoro -n-decanoic acid ( PFDA ), is a ten carbon straight-chain perfluorinated ...following: 2mg, 20mg or 50mg PFDA /kg body weight; single injection; animals sacrificed on day 8 of exposure 150mg PFOA /kg ( perfluorooctanoate , PFDA’s...from the following treatments: (I) control; (II) 50mg/kg PFDA ; (III) 100mg/kg PFOA ; and (IV) 400mg/kg clofibrate. Panels V and VI are nitrocellulose

Characterization and Fate of Gun and Rocket Propellant Residues on Testing and Training Ranges: Interim Report 2

DTIC Science & Technology

2010-12-01

compounds and stabiliz- ers in more conventional energetic formulations such as nitrocellulose (NC)-based propellants. Assessing the deposition...were opened and spread out to dry on aluminum foil covered trays to dry at room temperature. The dried material was then sieved under a hood with a...filtrate generated were tracked. The filters were placed in a labeled jar and set out to dry . After drying , the jars were sealed and refrigerated

Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon.

PubMed

Patil, R; Shankar, K M; Kumar, B T N; Kulkarni, A; Patil, P; Moger, N

2013-09-01

A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL(-1) crude WSSV protein, respectively. The FTA could be completed in 8-10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4-8 °C. The FTA is available as a rapid test kit called 'RapiDot' for the early detection of WSSV under field conditions. © 2013 John Wiley & Sons Ltd.

Impact of different-sized laminar air flow versus no laminar air flow on bacterial counts in the operating room during orthopedic surgery.

PubMed

Diab-Elschahawi, Magda; Berger, Jutta; Blacky, Alexander; Kimberger, Oliver; Oguz, Ruken; Kuelpmann, Ruediger; Kramer, Axel; Assadian, Ojan

2011-09-01

This study investigated the influence of the size of unidirectional ceiling distribution systems on counts of viable microorganisms recovered at defined sites in operating room (ORs) and on instrument tables during orthopedic surgery. We compared bacterial sedimentation during 80 orthopedic surgeries. A total of 19 surgeries were performed in ORs with a large (518 cm × 380 cm) unidirectional ceiling distribution (colloquially known as laminar air flow [LAF]) ventilation system, 21 procedures in ORs with a small (380 cm × 120 cm) LAF system, and 40 procedures in ORs with no LAF system. Bacterial sedimentation was evaluated using both settle plates and nitrocellulose membranes. Multivariate linear regression analysis revealed that the colony-forming unit count on nitrocellulose membranes positioned on the instrument table was significantly associated only with the size of the unidirectional LAF distribution system (P < .001), not with the duration of the surgical intervention (P = .753) or with the number of persons present during the surgical intervention (P = .291). Our findings indicate that simply having an LAF ventilation system in place will not provide bacteria-free conditions at the surgical site and on the instrument table. In view of the limited number of procedures studied, our findings require confirmation and further investigations on the ideal, but affordable, size of LAF ventilation systems. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

[Optimization of the immunoelectrophoresis technic (western blot) for the confirmation of human immunodeficiency virus infection (HIV) in Panama].

PubMed

Pascale, J M; de Austin, E; de Moreno, N O; Ledezma, C; de Márquez, E; Blanco, R; Quiroz, E; Calzada, J; Vincensini, A R; de Martin, M C

1995-01-01

The purpose of this study is to report the results of the authors' investigation to apply the western blot technique (WB UP-LCS) in the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection. To do this, the authors separated the proteins of the HIV-1 virus by electrophoresis, based on their molecular weight, in poliacilamide gel with SDS (SDS-PAGE) during 3 hours at 200 volts. Then they electrotransferred these proteins to nitrocellulose paper during four hours at 200 milliamperes, with the aid of external cooling. The nitrocellulose strips were evaluated considering the incubation time (1 and 16 hours), two conjugates (human anti IgG with Peroxidase and human anti IgG Biotin plus Streptatividine with Peroxidase) and two dilutions of the patients' sera (1/50 and 1/100). Based on their results the Authors conclude that, in the first place, the optimal conditions for the test include a dilution of 1/100 of the patients serum, incubation of the serum for 16 hours and the use of the conjugate of anti human IgG with Biotin and Streptavidine with Peroxidase; secondary, that the immunologic reactivity against proteins p24 and gp 160/120 is the most important diagnostic criterion for the confirmation of infection with HIV-1 and that they obtained a diagnostic correlation of 100% at a cost which was 5 to 7 times less than that of the commercial system.

Immunoblotting assays for keratan sulfate.

PubMed

Yoon, Jung Hae; Brooks, Randolph; Halper, Jaroslava

2002-07-15

The detection of microquantities of glycosaminoglycans (GAGs) in biological samples has been hampered by the lack of sensitive methods. In this paper we describe the modification and development of three sensitive assays capable of detecting nanogram quantities of GAGs in biological samples. The first assay detects total GAGs. It is a modified Alcian blue dye precipitation assay in which the dye binds to the negatively charged GAGs in CsCl-fractionated extracts from chicken tendons. This assay compares favorably with the widely used uronic acid assay in terms of its sensitivity and ability to detect all classes of GAGs, including keratan sulfate (KS). Two other assays, dot-blotting and immunoblotting, detect KS in complex mixtures and can be easily adapted for the detection of other GAGs. Both take advantage of binding of carboxyl and sulfate groups of GAGs to trivalent neodymium. In dot-blotting, samples were directly blotted onto nitrocellulose membrane soaked in Nd(2)(SO(4))(3) buffer, and KS was detected with the monoclonal anti-KS 5-D-4 antibody and an avidin-biotin complex detection system. In immunoblotting, the samples were first separated in 28% polyacrylamide gels, transferred onto a Nd(2)(SO(4))(3)-soaked nitrocellulose membrane using a phosphate buffer system, and stained and developed using the same protocol as in dot-blotting. Whereas dot-blotting allows the use of very low quantities of samples because of its high sensitivity (lower detection limit was 5 ng), immunoblotting provides more specificity.

Mammalian Toxicity of Munition Compounds. Phase 1. Acute Oral Toxicity Primary Skin and Eye Irritation, Dermal Sensitization, and Disposition and Metabolism

DTIC Science & Technology

1975-07-22

Mononitroglycerins Nitrocellulose White Phosphor 20. ABSTRACT (C^nllnu eery and Identify by block number) 0D | jSJ’TJ M73 EDITION OF I NOV 6» IS...White Phosphorus 2. Synthesis of DNGs and MNGs 3. Analysis of Chemicals . C. Acute LD D. Primary Skin and Eye Irritation E. Dermal...by the Radford Army Ammunition Plant (Radford, Virginia). 2. Synthesis of DNGs and MNGs a. 1,2-DNG: to that of Dunstan et al.— The sample was

Cosmic ray particles with different LET values under various thicknesses of shielding in low altitude orbits: Calculations and Cosmos-2044 measurements

NASA Technical Reports Server (NTRS)

Benton, E. V.; Frank, A. L.; Benton, E. R.; Marenny, A. M.; Nymmik, R. A.; Suslov, A. A.

1995-01-01

Fluxes of cosmic ray particles with different LET values were measured on board the COSMOS-2044 biosatellite under various thicknesses of shielding by stacks of CR-39 and nitrocellulose plastic nuclear track detectors (mounted outside the satellite). The component composition of the particles detected under shieldings of 0.1-2.5 g cm(exp -2) is verified by comparing experimental data with the results of model simulations of the fluxes of galactic cosmic ray particles and of radiation belt protons.

Nonelectrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens to obtain 12 immunoblots.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

Protein blotting is an invaluable technique in immunology to detect and characterize proteins of low abundance. Proteins resolved on sodium dodecyl sulfate (SDS) polyacrylamide gels are normally transferred electrophoretically to adsorbent membranes such as nitrocellulose or polyvinylidene diflouride membranes. Here, we describe the nonelectrophroretic transfer of the Ro 60 (or SSA) autoantigen, 220- and 240-kD spectrin antigens, and prestained molecular weight standards from SDS polyacrylamide gels to obtain up to 12 immunoblots from a single gel and multiple sera.

Protein blotting protocol for beginners.

PubMed

Petrasovits, Lars A

2014-01-01

The transfer and immobilization of biological macromolecules onto solid nitrocellulose or nylon (polyvinylidene difluoride (PVDF)) membranes subsequently followed by specific detection is referred to as blotting. DNA blots are called Southerns after the inventor of the technique, Edwin Southern. By analogy, RNA blots are referred to as northerns and protein blots as westerns (Burnette, Anal Biochem 112:195-203, 1981). With few exceptions, western blotting involves five steps, namely, sample collection, preparation, separation, immobilization, and detection. In this chapter, protocols for the entire process from sample collection to detection are described.

Mammalian Toxicity of Munitions Compounds. Phase III. Effects of Life-Time Exposure. Part III. Nitrocellulose.

DTIC Science & Technology

1980-01-01

male No. 52-25 injured his left hind leg on the metal bench in the run. High dose female No. 54-38 had a rectal prolapse in Month 24. These dogs ...studied in dogs , rats and mice. A cotton control (fed 10% cotton linters) was included with each species. Ancillary studies included cyto- genetic...increase in feed consumption. No other effects were seen in dogs . In rats and mice, those fed 10% fiber (NC or linters) had decreased DO , 1473 EoTIOM OF’ t

Hepatocyte Growth Factor Inhibits Apoptosis by the Profibrotic Factor Angiotensin II via Extracellular Signal-regulated Kinase 1/2 in Endothelial Cells and Tissue Explants

DTIC Science & Technology

2010-12-01

were subjected to SDS- polyacryl - amide gel electrophoresis and electroblotted onto nitrocellulose membrane. Membranes were blocked with 5% bovine...1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g...buffer [50 mM Tris-HCl, 1 mM EGTA, 1% (wt/vol) CHAPS, 10% glycerol, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride

Biomarkers in the Detection of Prostate Cancer in African Americans

DTIC Science & Technology

2013-09-01

externalized 189  phosphatidylserine,  milk   fat  globule‐E8/lactoferrin (MFG‐E8), CD80, CD86, CD96, Rab‐5b and 190  MHC class I and MHC class II complexes (7...aggressive features of PCas. Initially, proteins , mRNA and DNA will be extracted from nitrocellulose blots of biopsies of the prostate which are being...Similarly, proteins associated with racial differences and/or aggressiveness will be identified by liquid chromatography mass spectrometry (LCMS) and by

Final Technical Report ONT/ASEE (Office of Naval Technology/American Society for Engineering Education) Postdoctoral Fellow on Contract N00014-83-D-0689.

DTIC Science & Technology

1988-02-19

phosphatase with BCIP/NBT appears to produce the most clearly visible spot on the membrane. The spot is a purple-blue color, but one must orient the membrane...phosphatase-labeled antibodies. With any of these reagents, we can produce a discrete and clearly visible spot on nitrocellulose membranes. The intensity of...substrate systems are used; however, one has to orient the membrane towards the light in such a manner that the spot is clearly visible . We have found

Application of poly(amidoamine) dendrimers for use in bionanomotor systems.

PubMed

Kolli, Madhukar B; Day, B Scott; Takatsuki, Hideyo; Nalabotu, Siva K; Rice, Kevin M; Kohama, Kazuhiro; Gadde, Murali K; Kakarla, Sunil K; Katta, Anjaiah; Blough, Eric R

2010-05-04

The study and utilization of bionanomotors represents a rapid and progressing field of nanobiotechnology. Here, we demonstrate that poly(amidoamine) (PAMAM) dendrimers are capable of supporting heavy meromyosin dependent actin motility of similar quality to that observed using nitrocellulose, and that microcontact printing of PAMAM dendrimers can be exploited to produce tracks of active myosin motors leading to the restricted motion of actin filaments across a patterned surface. These data suggest that the use of dendrimer surfaces will increase the applicability of using protein biomolecular motors for nanotechnological applications.

Characterization of a Shiga-like Toxin Converting Phage from an Escherichia coli Strain Responsible for Hemorrhagic Colitis in Humans

DTIC Science & Technology

1985-04-30

paper (Whatman, England) was placed in a large baking dish filled with 20X SSPE (salt-sodium phosphate-EDTA) (Maniatis, 1982) and air bubbles trapped...avoid air bubbles between the 3 MM paper and the gel). A nitrocellulose filter moistened with 20X SSPE was placed on top of the gel and air bubbles were...proceed for 24 hours, the positions were marked with a pen. The filter was then soaked in 5X SSPE 27 for 10 minutes, dried at room temperature, and

Cosmic ray particles with different LET values under various thicknesses of shielding in low altitude orbits: calculations and Cosmos-2044 measurements

NASA Technical Reports Server (NTRS)

Marenny, A. M.; Nymmik, R. A.; Suslov, A. A.; Benton, E. V.; Frank, A. L.; Benton, E. R.

1992-01-01

Fluxes of cosmic ray particles with different LET values were measured on board the Cosmos-2044 biosatellite under various thicknesses of shielding by stacks of CR-39 and nitrocellulose plastic nuclear track detectors (mounted outside the satellite). The component composition of the particles detected under shieldings of 0.1-2.5 g cm-2 is verified by comparing experimental data with the results of model simulations of the fluxes of galactic cosmic ray particles and of radiation belt protons.

Other notable protein blotting methods: a brief review.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

A brief review of other notable protein blotting methods.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

Human induced pluripotent stem cell-derived beating cardiac tissues on paper.

PubMed

Wang, Li; Xu, Cong; Zhu, Yujuan; Yu, Yue; Sun, Ning; Zhang, Xiaoqing; Feng, Ke; Qin, Jianhua

2015-11-21

There is a growing interest in using paper as a biomaterial scaffold for cell-based applications. In this study, we made the first attempt to fabricate a paper-based array for the culture, proliferation, and direct differentiation of human induced pluripotent stem cells (hiPSCs) into functional beating cardiac tissues and create "a beating heart on paper." This array was simply constructed by binding a cured multi-well polydimethylsiloxane (PDMS) mold with common, commercially available paper substrates. Three types of paper material (print paper, chromatography paper and nitrocellulose membrane) were tested for adhesion, proliferation and differentiation of human-derived iPSCs. We found that hiPSCs grew well on these paper substrates, presenting a three-dimensional (3D)-like morphology with a pluripotent property. The direct differentiation of human iPSCs into functional cardiac tissues on paper was also achieved using our modified differentiation approach. The cardiac tissue retained its functional activities on the coated print paper and chromatography paper with a beating frequency of 40-70 beats per min for up to three months. Interestingly, human iPSCs could be differentiated into retinal pigment epithelium on nitrocellulose membrane under the conditions of cardiac-specific induction, indicating the potential roles of material properties and mechanical cues that are involved in regulating stem cell differentiation. Taken together, these results suggest that different grades of paper could offer great opportunities as bioactive, low-cost, and 3D in vitro platforms for stem cell-based high-throughput drug testing at the tissue/organ level and for tissue engineering applications.

Activation thermodynamics of virus adsorption to solids.

PubMed Central

Preston, D R; Farrah, S R

1988-01-01

The kinetics of bacteriophage MS2, T2, and f2 adsorption to powdered nitrocellulose and disrupted Seitz S1 filters at pH 7 were determined as a function of temperature. Data from these studies were combined with data produced in a previous study on MS2 adsorption to clay by Stagg et al. (Appl. Environ. Microbiol. 33:385-391, 1977). These workers studied the adsorption of MS2 to bentonite clay as a function of temperature. Data from both this previous study and the current one were used to calculate the thermodynamic parameters of virus adsorption. The results show that adsorption of bacteriophages to the solids tested is a physical process (energy of activation, less than 40 kcal [168 J]/mol) rather than a chemical process (energy of activation, greater than 40 kcal/mol). The free energy of activation showed a high negative correlation (r = -0.904, r2 = 0.817) with the percentage of virus adsorption to the solids tested. The energy of activation was highly negatively correlated with the percentage of virus adsorption to nitrocellulose and clay (r = -0.913, r2 = 0.834) but poorly correlated with the percentage of virus adsorption to disrupted Seitz S1 filters (r = -0.348, r2 = 0.121). In general, under conditions in which the percentage of virus adsorption was low, the energy of activation, the free energy of activation, and the entropy of activation were high. Increasing the percentage of virus adsorbed by changing the adsorbing conditions or changing the adsorbing solid decreased the energy of activation, the free energy of activation, and the entropy of activation. PMID:3214152

Adaptable Detection Strategies in Membrane-Based Immunoassays: Calibration-Free Quantitation with Surface-Enhanced Raman Scattering Readout.

PubMed

Skuratovsky, Aleksander; Soto, Robert J; Porter, Marc D

2018-06-19

This paper presents a method for immunometric biomarker quantitation that uses standard flow-through assay reagents and obviates the need for constructing a calibration curve. The approach relies on a nitrocellulose immunoassay substrate with multiple physical addresses for analyte capture, each modified with different amounts of an analyte-specific capture antibody. As such, each address generates a distinctly different readout signal that is proportional to the analyte concentration in the sample. To establish the feasibility of this concept, equations derived from antibody-antigen binding equilibrium were first applied in modeling experiments. Next, nitrocellulose membranes with multiple capture antibody addresses were fabricated for detection of a model analyte, human Immunoglobulin G (hIgG), by a heterogeneous sandwich immunoassay using antibody-modified gold nanoparticles (AuNPs) as the immunolabel. Counting the number of colored capture addresses visible to the unassisted eye enabled semiquantitative hIgG determination. We then demonstrated that, by leveraging the localized surface plasmon resonance of the AuNPs, surface-enhanced Raman spectroscopy (SERS) can be used for quantitative readout. By comparing the SERS signal intensities from each capture address with values predicted using immunoassay equilibrium theory, the concentration of hIgG can be determined (∼30% average absolute deviation) without reference to a calibration curve. This work also demonstrates the ability to manipulate the dynamic range of the assay over ∼4 orders of magnitude (from 2 ng mL -1 to 10 μg mL -1 ). The potential prospects in applying this concept to point-of-need diagnostics are also discussed.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

PubMed Central

Watts, T H; Worobec, E A; Paranchych, W

1982-01-01

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain. Images PMID:6813311

Characterization of a Shiga-Like Toxin Converting Phage Isolated from an Escherichia coli Strain Responsible for Hemorrhagic Colitis in Humans

DTIC Science & Technology

1985-01-01

filled with 20X SSPE (salt-sodium phosphate-EDTA) (Maniatis, 1982) and air bubbles trapped under the 3 ýM paper were pressed out with a glass rod. Next...nitrocellulose filter moist ned with 20X SSPE was placed on top of the gel and air bubbles were again pressed out with a glass rod. Two pieces of 3 M paper were...then soaked in 5X SSPE 27 for 10 minutes, dried at room temperature, and baked for 2 hours at 800 C under vacuum (Maniatis et al., 1982

Surface coating influence on elastic properties of spruce wood by means of holographic vibration mode visualization

NASA Astrophysics Data System (ADS)

Bongova, M.; Urgela, Stanislav

1999-07-01

Physicoacoustical properties of wood influenced by surface coating are studied by modal analysis. Resonant spruce plates were coated by stain, nitrocellulose varnish, special violin paint and shellac. The modal testing was performed by electronic speckle pattern interferometry. For this purpose, equipment called VIBROVIZER was used. The collected values of physicoacoustical characteristics (density, Young's modulus, acoustic constant) were compared using the graphic plots of data. The 3D plots help to evaluate wooden plates from a viewpoint of the quality control. This fact offers new opportunity for musical instrument manufacturers.

Invisible Ink Marking in ECL Membrane Assays.

PubMed

Kurien, Biji T

2015-01-01

Invisible ink and writing secret messages have been part of man's fantasy, having proven useful in clandestine and high sensitivity areas. Security inks, made up of invisible materials that give printed, anti-photocopy images capable of being read only under special environments, have become important. An ink formulation based on silicon(IV) 2,3-naphthalocyanine bis(trihexylsilyloxide) as colorant, invisible to the naked eye but infrared readable, has been described earlier. Biometric DNA ink has also been developed for security authentication. In lighter vein, many budding scientists and others have often experimented with writing secret messages on paper, either for purposes of fun or actually sending secret messages to friends. It involved the use of lemon juice, milk, or other solutions that could be used with a dip pen, brush, or a fountain pen to write invisible messages on a blank white paper. Words turn up as magic when the paper is exposed to heat in one form or the other. Here, we attempt to end this book on a slightly humorous note by showing that invisible messages can be written on nitrocellulose membranes (but not on polyvinylidene difluoride membranes) using an appropriately diluted horse radish peroxidase/alkaline phosphatase anti-IgG conjugate (rabbit, mouse, or human anti-IgG). The message is written on the membrane, preferably with a fountain pen, and the membrane is allowed to dry. Regular detection with enhanced chemiluminescence plus or nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate systems is used to unravel the secret message. In addition, this method could be used to mark nitrocellulose membranes for orientation purposes using ECL detection system and thus can eliminate the use of autoradiography pens.

Enhanced alkaline hydrolysis and biodegradability studies of nitrocellulose-bearing missile propellant

NASA Technical Reports Server (NTRS)

Sidhoum, Mohammed; Christodoulatos, Christos; Su, Tsan-Liang; Redis, Mercurios

1995-01-01

Large amounts of energetic materials which have been accumulated over the years in various manufacturing and military installations must be disposed of in an environmentally sound manner. Historically, the method of choice for destruction of obsolete or aging energetic materials has been open burning or open detonation (OB/OD). This destruction approach has become undesirable due to air pollution problems. Therefore, there is a need for new technologies which will effectively and economically deal with the disposal of energetic materials. Along those lines, we have investigated a chemical/biological process for the safe destruction and disposal of a double base solid rocket propellant (AHH), which was used in several 8 inch projectile systems. The solid propellant is made of nitrocellulose and nitroglycerin as energetic components, two lead salts which act as ballistic modifiers, triacetin as a plasticizer and 2-Nitrodiphenylamine (2-NDPA) as a stabilizer. A process train is being developed to convert the organic components of the propellant to biodegradable products and remove the lead from the process stream. The solid propellant is first hydrolyzed through an enhanced alkaline hydrolysis process step. Following lead removal and neutralization, the digested liquor rich in nitrates and nitrites is found to be easily biodegradable. The digestion rate of the intact ground propellant as well as the release of nitrite and nitrate groups were substantially increased when ultrasound were supplied to the alkaline reaction medium compared to the conventional alkaline hydrolysis. The effects of reaction time, temperature, sodium hydroxide concentration and other relevant parameters on the digestion efficiency and biodegradability have been studied. The present work indicates that the AHH propellant can be disposed of safely with a combination of physiochemical and biological processes.

Development of an immunochromatographic strip for simple detection of penicillin-binding protein 2'.

PubMed

Matsui, Hidehito; Hanaki, Hideaki; Inoue, Megumi; Akama, Hiroyuki; Nakae, Taiji; Sunakawa, Keisuke; Omura, Satoshi

2011-02-01

Infections with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2' (PBP2' or PBP2a), which shows low affinity to β-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2' within about 20 min. Anti-PBP2' monoclonal antibodies were produced by a hybridoma of recombinant PBP2' (rPBP2')-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2' of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2', and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2'-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2' or 2.8 × 10(5) to 1.7 × 10(7) CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and a Candida albicans strain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2' gene, mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2'-producing cells in clinical laboratories.

An Implantable Transparent Conductive Film with Water Resistance and Ultrabendability for Electronic Devices.

PubMed

Song, Youngjun; Kim, Sejung; Heller, Michael J

2017-12-06

Recently, instead of indium tin oxide, the random mesh pattern of metallic nanowires for flexible transparent conducting electrodes (FTCEs) has received a great amount of interest due to its flexibility, low resistance, reasonable price, and compliant processes. Mostly, nanowires for FTCEs are fabricated by spray or mayer coating methods. However, the metallic nanowire layer of FTCEs, which is fabricated by these methods, has a spiked surface roughness and low junction contact between the nanowires that lead to their high sheet resistance value. Also, the nanowires on FTCEs are easy to peel-off through exterior forces such as bending, twisting, or contact. To solve these problems, we demonstrate novel methods through which silver nanowires (AgNWs) are deposited onto a nanosize porous nitrocellulose (NC) substrate by electrophoretic deposition (EPD) and an opaque and porous substrate. Respectively, through dimethyl sulfoxide treatment, AgNWs on NC (AgNW/NC) is changed to the transparent and nonporous FTCEs. This enhances the junction contact of the AgNWs by EPD and also allows a permanent attachment of AgNWs onto the substrate. To show the mechanical strength of the AgNWs on the transparent nitrocellulose (AgNW/TNC), it is tested by applying diverse mechanical stress, such as a binding test (3M peel-off), compressing, bending, twisting, and folding. Next, we demonstrate that AgNW/TNC can be effectively implanted onto normal newspapers and papers. As paper electronics, light-emitting diodes, which are laminated onto paper, are successfully operated through a basic AgNW/TNC strip circuit. Finally, it is demonstrated that AgNW/TNC and AgNW/TNC on paper are water resistant for 15 min due to the insulation properties of the nonporous substrate.

Antigen detection based on background fluorescence quenching immunochromatographic assay.

PubMed

Chen, Xiangjun; Xu, Yangyang; Yu, Jinsheng; Li, Jiutong; Zhou, Xuelei; Wu, Chuanyong; Ji, Qiuliang; Ren, Yuan; Wang, Liqun; Huang, Zhengyi; Zhuang, Hanling; Piao, Long; Head, Richard; Wang, Yajie; Lou, Jiatao

2014-09-02

Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100ngmL(-1) of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1ngmL(-1). Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r=0.98, p<0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Human autoantibodies against a desmosomal core protein in pemphigus foliaceus

PubMed Central

1984-01-01

Pemphigus foliaceus (PF) is a human autoimmune disease in which antibodies are directed against the cell surface of epidermal cells with resultant blister formation. The histopathology of these blisters indicates that cells have detached from each other, and electron microscopy of early blisters shows diminished numbers, to complete loss, of desmosomes as well as abnormalities of the tonofilament- desmosome complex. In this study we demonstrate that autoantibodies from certain PF patients bind to a desmosomal core glycoprotein called desmoglein (DG) I. Proteins in extracts of normal human epidermis were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), then transferred to nitrocellulose or 2- aminophenylthioether paper for immunoperoxidase staining. Results of these immunoblots indicated that sera from 6 of 13 PF patients specifically and intensely stained an approximately 160,000 mol wt polypeptide, "PF antigen". Such staining was not seen with normal human sera or sera from patients with pemphigus vulgaris or bullous pemphigoid, two autoimmune blistering skin diseases that are clinically, histologically, and immunochemically distinct from PF. However, rabbit antiserum directed against DGI, that was isolated from bovine muzzle desmosomes, stained a polypeptide band which co-migrated with PF antigen. Furthermore, when proteins from extracts of normal human epidermis were electrophoresed in two dimensions (isoelectric focusing, then SDS-PAGE) before transfer to nitrocellulose for immunoperoxidase staining, PF antibodies and antibodies to DGI stained identical spots. Finally, PF sera as well as PF IgG that was affinity purified with PF antigen from normal human epidermis, both selectively bound to DGI extracted from bovine muzzle desmosomes. These studies demonstrate that the human autoantibodies from certain patients with PF, a disease of epidermal cell adhesion, are directed against a desmosomal core protein. PMID:6491602

UNIT 10.7 Electroblotting from Polyacrylamide Gels

PubMed Central

Goldman, Aaron; Speicher, David W.

2015-01-01

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications. PMID:26521711

Electroblotting from Polyacrylamide Gels.

PubMed

Goldman, Aaron; Ursitti, Jeanine A; Mozdzanowski, Jacek; Speicher, David W

2015-11-02

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications. Copyright © 2015 John Wiley & Sons, Inc.

Restriction fragment length polymorphism among Israeli Holstein-Friesian dairy bulls.

PubMed

Beckmann, J S; Kashi, Y; Hallerman, E M; Nave, A; Soller, M

1986-01-01

Israeli Holstein-Friesian dairy bulls were screened for restriction fragment length polymorphisms by hybridizing cloned DNA probes for bovine growth hormone, for chymosin, and for rat muscle beta-actin to restriction endonuclease-digested DNA immobilized on nitrocellulose filters. The population proved to be polymorphic at the growth hormone locus, with evidence consistent with the phenotypes being inherited in allelic fashion. A low level of polymorphism was also observed at one of the beta-actin gene family loci. The chymosin locus was monomorphic with the restriction enzymes utilized. The results illustrate the power of restriction fragment length polymorphism methodology in visualizing genetic variability in dairy cattle populations.

The Feasibility of Concentrating/Separating Dilute Nitrocellulose Acid Wastewaters by Reverse Osmosis

DTIC Science & Technology

1974-11-01

HO Membrane 18 I ■ ■■ mmi 11 TABLES Table 3 4 8 9 Title Page Acid Cumpositiun of Wastewater from Mtrocollulosc Area - Boiling Tub Pit 2...8217 • # • • 9600 9100 ** 1 I ♦ # • • 8800 ** «• 16 «« <moo 9000 11,000 #« 18 *• *« «* 14.000 • • 20 ** liVOOO 10,500 •* ** »i ** • « 17,000 ** 21...14 ** •* 2640 ** •* 16 *• 2750 2600 2840 •♦ 18 •* •• •• 2790 •• 20 •• 2860 2210 •• ♦• 22 •» # # 2770

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

Yeager, John David; Watkins, Erik Benjamin; Duque, Amanda Lynn

Thermal ignition via self-heating (cook-off) of cyclotetramethylene-tetranitramine (HMX)-containing plastic-bonded explosives (PBXs) is driven by the β → δ phase transition in the HMX, which is affected if not dominated by microstructure. Here, we studied the HMX-binder interface and phase transition for several variations of PBX 9404 (HMX with plasticized nitrocellulose [NC] binder). Neutron reflectometry was used to examine the interface under several conditions—pristine, after aging, and after thermal treatment. The initial interfacial structure depended on the plasticizer, but the interface homogenized over time. Thermal and optical analyses showed that all formulated materials had higher transition temperatures than neat HMX. Thismore » effect increased with NC content.« less

Western Blotting of the Endocannabinoid System.

PubMed

Wager-Miller, Jim; Mackie, Ken

2016-01-01

Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

Computations on Internal Blast from Titanium-Cased Charges in Air

DTIC Science & Technology

1984-07-01

formation and considerable temperature- dependent polymorphic transition . Oxygen is soluble in titanium metal to the extent of about 25 atom-percent, yielding...15 CSH8N4O1 2 NC Nitrocellulose, 13.3% N; -812.9 +3 C6H7N2.5010 HMX symn-Cyclotetramnethylene- + 77.4 0 tetranitramine, C4HSNBO8 Pentolite 50% PETN, 50...TNT; -97.9 -5 C6.1 6H6 .2 5N3.41085. Comp 8 65% RDX , 35% TNT; + 11.47 -9 C1 .96H2. 53N2.2202.68 TNT 2,4,6-Trinitrotoluene, +30.7 -25 p C7R 5N306 N2

Development of nanoparticle applications in cell imaging, bioassay and reactive oxygen species detection based on surface-enhanced raman spectroscopy

NASA Astrophysics Data System (ADS)

Huang, Yiming

Surface-enhanced Raman scattering (SERS) has been developed over forty years with a wide variety of applications. Signals enhanced from single molecule absorbed on the surface of metallic nanoparticles can be up to 14-order-of-magnitude. This is due to the resonance between the optical field and surface plasmon of the metal substrate. Nanoshells have been chosen as substrates since they do not need to pre-aggregate due to their tunable optical property. We developed Raman imaging system by incorporating functionalized nanoshells, cells and SERS. Nanoshells have been coated with different self-assembled monolayers containing poly(ethylene glycol) (PEG) molecules. Probes have been designed by coating nanoshells with Raman active PEG molecules and delivered into macrophage cells. The imaging technique requires less preparation and provides the information of nanoshells in semi-quantitative way in vitro. We developed half-sandwich bioassay by detecting low volume of antigens on nitrocellulose membrane, detected by SERS. Antibodies were grafted to the surface of nanoshells and were conjugated to the antigens on the nitrocellulose membrane for detection. Raman active PEGs were conjugated onto the metal substrate for recognition and quantification. The benefits of this assay are that it is faster, easier to execute and requires less volume of antigen to conjugate onto the substrate. We also developed reactive oxygen species (ROS) sensors by incubating PEGs and either 4-nitrobenzenethiol (4-NBT) or 4-mercaptophenol (4-MP) on the surface of nanoshells. By analyzing the changes of SERS spectrum, the production of hydroxyl radicals produced in the Fenton reaction can be tracked in low concentrations. The sensors were designed to track ROS production within cells when they are under oxidative stress. The methods developed in this thesis are versatile, and can be broadly applied to the study of different subtracts, such as gold colloid.

Effect of mobile unidirectional air flow unit on microbial contamination of air in standard urologic procedures.

PubMed

Ferretti, Stefania; Pasquarella, Cesira; Fornia, Samanta; Saccani, Elisa; Signorelli, Carlo; Vitali, Pietro; Sansebastiano, Giuliano Ezio

2009-12-01

Infection is one of the most feared complications of surgery. New instrumentation is being developed to reduce deposition of bacteria. We investigated 45 major surgical procedures (21 radical nephrectomies [RN] and 24 radical retropubic prostatectomies [RRP]) in our urology department during 2007. In about one-half of the interventions, an ultraclean air flow mobile (UAF) unit was used. Bacterial sedimentation was evaluated by nitrocellulose membranes placed on the instrument tray and by settle plates positioned at four points in the operating room. In 27 operations, an additional membrane was located near the incision. Bacterial counts on the nitrocellulose membranes during RN were 230 colony-forming units (cfu)/m(2)/h with the UAF unit and 2,254 cfu/m(2)/h without the unit (p = 0.001). During RRP, the values were 288 cfu/m(2)/h and 3,126 cfu/m(2)/h respectively (p = 0.001). The membrane placed near the incision during RN showed a microbial count of 1,235 cfu/m(2)/h with the UAF unit and 5,093 cfu/m(2)/h without the unit (p = 0.002); during RRP, the values were 1,845 cfu/m(2)/h and 3,790 cfu/m(2)/h, respectively (difference not significant). Bacterial contamination detected by settle plates during RN showed a mean value of 2,273 cfu/m(2)/h when the UAF unit was used and 2,054 cfu/m(2)/h without the unit; during RRP, the values were 2,332 cfu/m(2)/h and 2,629 cfu/m(2)/h with and without the UAF unit, respectively (NS). No statistically significant differences were detected in the clinical data registered in patients operated on under standard conditions and while the UAF unit was functioning. The UAF appears able to reduce microbial contamination at the operating table, reaching a bacterial number obtained in ultraclean operating theatres.

A rapid two dot filter assay for the detection of E. coli O157 in water samples.

PubMed

Kamma, Sujatha; Tang, Lily; Leung, Kelvin; Ashton, Edie; Newman, Norman; Suresh, Mavanur R

2008-07-31

E. coli O157:H7 is an enterohemorrhagic bacteria that cause deadly water-borne infections implicated in outbreaks of a wide spectrum of human gastrointestinal diseases. It is therefore important to have a rapid convenient, simple and sensitive range of detection of E. coli O157:H7. A new E. coli O157 MAb designated P124 was developed for ultrasensitive detection of E. coli O157 in water, apple juice and beef for routine use. A prototype filter dot assay was designed with anti-E. coli O157 MAb bound to 0.2 microm nitrocellulose filter disk as the capture antibody. A 100 ml water sample spiked with 1-50 CFU of E. coli O157 either in the presence or absence of other non-specific bacteria were filtered for capture of the pathogen on the antibody coated nitrocellulose disk. The detection of the pathogen was successfully accomplished by the same antibody both as a capture and detecting antibody as a homosandwich. In a non-enriched format, detection of E. coli was possible with a sensitivity of 2500 CFU/100 ml. Ultrasensitive detection of ~1 CFU/100 ml sample could be achieved by a prior pathogen enrichment step before the addition of the labeled antibody. The design of this diagnostic test is based on the common architecture of all bacteria, viruses and spores, namely the manifestation of repeat lipopolysaccharide epitopes on the surface. We have developed an easy-to-use two dot visual filter assay for translation into current water testing in public health laboratories to detect E. coli O157:H7. In a 5 h assay approximately 1 CFU and approximately 5 CFU of E. coli O157 could be detected in 100 ml of water or juice and lake samples respectively. This simple homosandwich enrichment strategy can also be used to detect low levels of other water-borne pathogens.

A test strip platform based on DNA-functionalized gold nanoparticles for on-site detection of mercury (II) ions.

PubMed

Guo, Zhiyong; Duan, Jing; Yang, Fei; Li, Min; Hao, Tingting; Wang, Sui; Wei, Danyi

2012-05-15

A test strip, based on DNA-functionalized gold nanoparticles for Hg(2+) detection, has been developed, optimized and validated. The developed colorimetric mercury sensor system exhibited a highly sensitive and selective response to mercury. The measurement principle is based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and streptavidin-biotin interaction. A biotin-labeled and thiolated DNA was immobilized on the gold nanoparticles (AuNPs) surface through a self-assembling method. Another thymine-rich DNA, which was introduced to form DNA duplexes on the AuNPs surface with thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination in the presence of Hg(2+), was immobilized on the nitrocellulose membrane as the test zone. When Hg(2+) ions were introduced into this system, they induced the two strands of DNA to intertwist by forming T-Hg(2+)-T bonds resulting in a red line at the test zone. The biotin-labeled and thiolated DNA-functionalized AuNPs could be captured by streptavidin which was immobilized on the nitrocellulose membrane as the control zone. Under optimized conditions, the detection limit for Hg(2+) was 3 nM, which is lower than the 10nM, maximum contaminant limit defined by the US Environmental Protection Agency (EPA) for drinking water. A parallel analysis of Hg(2+) in pool water samples using cold vapor atomic absorption spectrometry showed comparable results to those obtained from the strip test. Therefore, the results obtained in this study could be used as basic research for the development of Hg(2+) detection, and the method developed could be a potential on-site screening tool for the rapid detection of Hg(2+) in different water samples without special instrumentation. All experimental variables that influence the test strip response were optimized and reported. Copyright © 2012 Elsevier B.V. All rights reserved.

Development of a solvent processed insensitive propellant

NASA Technical Reports Server (NTRS)

Trask, R.; Costa, E.; Beardell, A. J.

1980-01-01

Two types of low vulnerability propellants are studied which are distinguished by whether the binder is a rubber, such as polyurethane or CTBN, or a plasticizable polymer such as ethyl cellulose or cellulose acetate. The former propellants are made by a partial cure extrusion process while the latter are made by the conventional solvent process. Emphasis is given to a cellulose binder (plasticizer) RDX composition. The type of binder used, the particle size of the RDX and the presence of small quantities of nitrocellulose in the solvent processed compositions have important influences on the mechanical and combustion characteristics of the propellant. The low temperature combustion is of particular concern because of potential breakup of the grains that can lead to instability.

Complexes of Nitrocellulose with Cupric Chloride,

DTIC Science & Technology

1985-11-01

4 Z I Vm 04 N-C-11 0soa -~~~ ii a Lid US 1 U C. U . i .ci OC S 0V- C C C 0 d i 0 41 v . 0i C u -s4 0- C .4 ~ tw aM 0i u U-JU I CU- w4 05 a.- ow US...la formation *d’un complexe et de la fraction pond&rale de CC par rapport A la NC, X , dltermin~ s au point de saturation. Le PCC est caractfristique de...59 s - 1. 3.5 Effect of X on the Rate of Complex Formation The variation of the ratio (ki/kf) with X is shown in Fig. 4 for sample 11 at C - 59 s

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

NASA Technical Reports Server (NTRS)

Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

1993-01-01

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

Mechanochemical Nitration of Aromatic Compounds

NASA Astrophysics Data System (ADS)

Lagoviyer, Oleg S.; Krishtopa, Larisa; Schoenitz, Mirko; Trivedi, Nirupam J.; Dreizin, Edward L.

2018-04-01

Nitration of organic compounds is necessary to produce many energetic materials, such as TNT and nitrocellulose. The conventional nitration process uses a mixture of concentrated sulfuric and nitric acids as nitrating agents and multiple solvents. The chemicals are corrosive and require special handling and disposal procedures. In this study, aromatic nitration has been achieved using solvent-free mechanochemical processing of environmentally benign precursors. Mononitrotoluene was synthesized by milling toluene with sodium nitrate and molybdenum trioxide as a Lewis acid catalyst. Several parameters affecting the desired product yield were identified and varied. A number of byproducts, i.e., dimers of toluene were also produced, but the selectivity was observed to increase with increasing mononitrotoluene yield. Both absolute mononitrotoluene yields and selectivity of its production increased with the increase in the energy transferred to the material from the milling tools.

A paper-based device for double-stranded DNA detection with Zif268

NASA Astrophysics Data System (ADS)

Zhang, Daohong

2017-05-01

Here, a small analytical device was fabricated on both nitrocellulose membrane and filter paper, for the detection of biotinylated double-stranded DNA (dsDNA) from 1 nM. Zif268 was utilized for capturing the target DNA, which was a zinc finger protein that recognized only a dsDNA with specific sequence. Therefore, this detection platform could be utilized for PCR result detection, with the well-designed primers (interpolate both biotin and Zif268 binding sequence). The result of the assay could be recorded by a camera-phone, and analyzed with software. The whole assay finished within 1 hour. Due to the easy fabrication, operation and disposal of this device, this method can be employed in point-of-care detection or on-site monitoring.

Performance evaluation for different sensing surface of BICELLs bio-transducers for dry eye biomarkers

NASA Astrophysics Data System (ADS)

Laguna, M. F.; Holgado, M.; Santamaría, B.; López, A.; Maigler, M.; Lavín, A.; de Vicente, J.; Soria, J.; Suarez, T.; Bardina, C.; Jara, M.; Sanza, F. J.; Casquel, R.; Otón, A.; Riesgo, T.

2015-03-01

Biophotonic Sensing Cells (BICELLs) are demonstrated to be an efficient technology for label-free biosensing and in concrete for evaluating dry eye diseases. The main advantage of BICELLs is its capability to be used by dropping directly a tear into the sensing surface without the need of complex microfluidics systems. Among this advantage, compact Point of Care read-out device is employed with the capability of evaluating different types of BICELLs packaged on Biochip-Kits that can be fabricated by using different sensing surfaces material. In this paper, we evaluate the performance of the combination of three sensing surface materials: (3-Glycidyloxypropyl) trimethoxysilane (GPTMS), SU-8 resist and Nitrocellulose (NC) for two different biomarkers relevant for dry eye diseases: PRDX-5 and ANXA-11.

[Evaluation of the total biological activity and allergenic composition of allergenic extracts].

PubMed

Lombardero, M; González, R; Duffort, O; Juan, F; Ayuso, R; Ventas, P; Cortés, C; Carreira, J

1986-01-01

In the present study, a complete procedure is presented in order to standardize allergenic extracts, the meaning of which is the measurement of the total allergenic activity and the determination of the allergenic composition. The measurement of the biological activity comprises 2 steps: Preparation of Reference Extracts and determination of their "in vivo" activity. Evaluation of the total allergenic activity of extracts for clinical use. Reference extracts were prepared from the main allergens and their "in vivo" biological activity was determined by a quantitative skin prick test in a sample of at least 30 allergic patients. By definition, the protein concentration of Reference Extract that produces, in the allergic population, a geometric mean wheal of 75 mm.2 has an activity of 100 biological units (BUs). The determination of the biological activity of a problem extract is made by RAST inhibition. The sample is compared with the corresponding Reference Extract by this technique and, from this comparison, it is possible to quantify the activity of the problem extract in biologic units (BUs) with clinical significance. Likewise, different techniques have been used to determine the allergenic composition of extracts. These techniques comprise 2 steps: Separation of the components of the extract. Identification of the components that bind specific human IgE. The separation of the components of the extract has been carried out by isoelectric focusing (IEF) and electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). In order to identify the allergenic components, an immunoblotting technique has been employed. The separated components in the IEF gel or SDS-PAGE gel are transferred to a nitrocellulose sheet and later on, this membrane is overlaid with a serum pool from allergic patients and a mouse monoclonal anti-human IgE, labelled with 125I. Finally, the autoradiography of the nitrocellulose membrane is obtained. In this way it is possible to compare the allergenic composition of an extract with the corresponding Reference Extract and so to employ for clinical use only those extracts with the right allergenic composition.

[GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains].

PubMed

Markina, O V; Alekseeva, L P; Telesmanich, N R; Chemisova, O S; Akulova, M V; Markin, N V

2011-05-01

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

Oxidation resistant slurry coating for carbon-based materials

NASA Technical Reports Server (NTRS)

Smialek, J. L.; Rybicki, G. C. (Inventor)

1985-01-01

An oxidation resistant coating is produced on carbon-base materials, and the same processing step effects an infiltration of the substrate with silicon containing material. The process comprises making a slurry of nickel and silicon powders in a nitrocellulose lacquer, spraying onto the graphite or carbon-carbon substrate, and sintering in vacuum to form a fused coating that wets and covers the surface as well as penetrates into the pores of the substrate. Optimum wetting and infiltration occurs in the range of Ni-60 w/o Si to Ni-90 w/o Si with deposited thicknesses of 25-100 mg/sq. cm. Sintering temperatures of about 1200 C to about 1400 C are used, depending on the melting point of the specific coating composition. The sintered coating results in Ni-Si intermetallic phases and SiC, both of which are highly oxidation resistant.

Conjugative plasmid in Corynebacterium flaccumfaciens subsp. oortii that confers resistance to arsenite, arsenate, and antimony(III)

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

Hendrick, C.A.; Haskins, W.P.; Vidaver, A.K.

1984-07-01

Gene transfer systems for phytopathogenic corynebacteria have not been reported previously. In this paper a conjugative 46-megadalton plasmid (pDG101) found in Corynebacterium flaccumfaciens subsp. oorii CO101 is described that mediates resistance to arsenite, arsenate, and antimony(III). Transfer of the plasmid from CO101 to four other strains from the C. flaccumfaciens group occurred between cells immobilized on nitrocellulose filters or on agar surfaces. Transconjugant strains expressed the same levels of metal resistance as the donor strain and were able to act as donor strains in subsequent matings. The physical presence of the plasmid was detected by agarose gel electrophoresis. Arsenite-sensitive derivativesmore » of the donor and transconjugant strains were obtained after heat treatment; these were cured of pDG101.« less

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Two Dimensional Polymer That Generates Nitric Oxide.

DOEpatents

McDonald, William F.; Koren, Amy B.

2005-10-04

A polymeric composition that generates nitric oxide and a process for rendering the surface of a substrate nonthrombogenic by applying a coating of the polymeric composition to the substrate are disclosed. The composition comprises: (1) a crosslinked chemical combination of (i) a polymer having amino group-containing side chains along a backbone forming the polymer, and (ii) a crosslinking agent containing functional groups capable of reacting with the amino groups; and (2) a plurality of nitric oxide generating functional groups associated with the crosslinked chemical combination. Once exposed to a physiological environment, the coating generates nitric oxide thereby inhibiting platelet aggregation. In one embodiment, the nitric oxide generating functional groups are provided by a nitrated compound (e.g., nitrocellulose) imbedded in the polymeric composition. In another embodiment, the nitric oxide generating functional groups comprise N2O2- groups covalently bonded to amino groups on the polymer.

Study of pipe thickness loss using a neutron radiography method

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

Mohamed, Abdul Aziz; Wahab, Aliff Amiru Bin; Yazid, Hafizal B.

2014-02-12

The purpose of this preliminary work is to study for thickness changes in objects using neutron radiography. In doing the project, the technique for the radiography was studied. The experiment was done at NUR-2 facility at TRIGA research reactor in Malaysian Nuclear Agency, Malaysia. Test samples of varying materials were used in this project. The samples were radiographed using direct technique. Radiographic images were recorded using Nitrocellulose film. The films obtained were digitized to processed and analyzed. Digital processing is done on the images using software Isee!. The images were processed to produce better image for analysis. The thickness changesmore » in the image were measured to be compared with real thickness of the objects. From the data collected, percentages difference between measured and real thickness are below than 2%. This is considerably very low variation from original values. Therefore, verifying the neutron radiography technique used in this project.« less

TATA Binding Protein Discriminates between Different Lesions on DNA, Resulting in a Transcription Decrease

PubMed Central

Coin, Frédéric; Frit, Philippe; Viollet, Benoit; Salles, Bernard; Egly, Jean-Marc

1998-01-01

DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position −29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage. PMID:9632775

Chlamydia trachomatis elementary bodies possess proteins which bind to eucaryotic cell membranes

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

Wenman, W.M.; Meuser, R.U.

1986-02-01

Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L/sub 2/ were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000- and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000- and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizingmore » activity. These data suggest that these putative chlamydial adhesions play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.« less

Draft genome sequence of Paenibacillus sp. EZ-K15 isolated from wastewater systems.

PubMed

Mohammed, Waleed S; Ziganshina, Elvira E; Shagimardanova, Elena I; Gogoleva, Natalia E; Ziganshin, Ayrat M

2017-12-12

Paenibacillus species, belonging to the family Paenibacillaceae, are able to survive for long periods under adverse environmental conditions. Several Paenibacillus species produce antimicrobial compounds and are capable of biodegradation of various contaminants; therefore, more investigations at the genomic level are necessary to improve our understanding of their ecology, genetics, as well as potential biotechnological applications. In the present study, we describe the draft genome sequence of Paenibacillus sp. EZ-K15 that was isolated from nitrocellulose-contaminated wastewater samples. The genome comprises 7,258,662 bp, with a G+C content of 48.6%. This whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PDHM00000000. Data demonstrated here can be used by other researchers working or studying in the field of whole genome analysis and application of Paenibacillus species in biotechnological processes.

High frequency lateral flow affinity assay using superparamagnetic nanoparticles

NASA Astrophysics Data System (ADS)

Lago-Cachón, D.; Rivas, M.; Martínez-García, J. C.; Oliveira-Rodríguez, M.; Blanco-López, M. C.; García, J. A.

2017-02-01

Lateral flow assay is one of the simplest and most extended techniques in medical diagnosis for point-of-care testing. Although it has been traditionally a positive/negative test, some work has been lately done to add quantitative abilities to lateral flow assay. One of the most successful strategies involves magnetic beads and magnetic sensors. Recently, a new technique of superparamagnetic nanoparticle detection has been reported, based on the increase of the impedance induced by the nanoparticles on a RF-current carrying copper conductor. This method requires no external magnetic field, which reduces the system complexity. In this work, nitrocellulose membranes have been installed on the sensor, and impedance measurements have been carried out during the sample diffusion by capillarity along the membrane. The impedance of the sensor changes because of the presence of magnetic nanoparticles. The results prove the potentiality of the method for point-of-care testing of biochemical substances and nanoparticle capillarity flow studies.

The quest for improved reproducibility in MALDI mass spectrometry.

PubMed

O'Rourke, Matthew B; Djordjevic, Steven P; Padula, Matthew P

2018-03-01

Reproducibility has been one of the biggest hurdles faced when attempting to develop quantitative protocols for MALDI mass spectrometry. The heterogeneous nature of sample recrystallization has made automated sample acquisition somewhat "hit and miss" with manual intervention needed to ensure that all sample spots have been analyzed. In this review, we explore the last 30 years of literature and anecdotal evidence that has attempted to address and improve reproducibility in MALDI MS. Though many methods have been attempted, we have discovered a significant publication history surrounding the use of nitrocellulose as a substrate to improve homogeneity of crystal formation and therefore reproducibility. We therefore propose that this is the most promising avenue of research for developing a comprehensive and universal preparation protocol for quantitative MALDI MS analysis. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:217-228, 2018. © 2016 Wiley Periodicals, Inc.

(Hydroxyproline-rich glycoprotein of the plant cell wall): Report on work from June 1987 to June 1988

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

Not Available

1988-01-01

In soybean seed costs the accumulation of the hydroxproline-rich glycoprotein extensin is regulated in a developmental and tissue-specific manner. The time course of appearance of extensin during seed development was studied by Western blot analysis and by immunogold-silver localization. Using these techniques extensin was first detected at 16 to 18 d after anthesis, increasing during development to high levels at 24 d after anthesis. Immunogold-silver localization of extensin in the seed coat showed marked depostion of the glycoprotein in the walls of palisade epidermal cells and hourglass cells. The immunolocalization of extensin in developing soybean seeds was also made bymore » a new technique - tissue printing on nitrocellulose paper. This technique shows that extensin is primarily localized in the seed coal, hilum, and vascular elements of the seed.« less

Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

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

Loper, J.C.; Chen, C.; Dey, C.R.

1993-01-01

Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodiummore » dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.« less

Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

PubMed

Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

2016-11-30

Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

A multiplex protein-free lateral flow assay for detection of microRNAs based on unmodified molecular beacons.

PubMed

Javani, Atefeh; Javadi-Zarnaghi, Fatemeh; Rasaee, Mohammad Javad

2017-11-15

Lateral flow assays (LFAs) have promising potentials for point-of-care applications. Recently, many LFAs have been reported that are based on hybridization of oligonucleotide strands. Mostly, biotinylated capture DNAs are immobilized on the surface of a nitrocellulose membrane via streptavidin interactions. During the assay, stable colorful complexes get formed that are visible by naked eyes. Here, we present an inexpensive and unique design of LFA that applies unmodified oligonucleotides at capture lines. The presented LFA do not utilize streptavidin or any other affinity protein. We employ structural switch of molecular beacons (MB) in combination with base stacking hybridization (BSH) phenomenon. The unique design of the reported LFA provided high selectivity for target oligonucleotides. We validated potential applications of the system for detection of DNA mimics of two microRNAs in multiplex assays. Copyright © 2017 Elsevier Inc. All rights reserved.

[Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection].

PubMed

Plekhanova, Iu V; Firsova, Iu E; Doronina, N V; Trotsenko, Iu A; Reshetilov, A N

2013-01-01

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6-8.8 mM) led to a change in the transistor's output signal, which was determined by the appearance of H+ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria--Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM 17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM 16--the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

MEMS-Based Solid Propellant Rocket Array Thruster

NASA Astrophysics Data System (ADS)

Tanaka, Shuji; Hosokawa, Ryuichiro; Tokudome, Shin-Ichiro; Hori, Keiichi; Saito, Hirobumi; Watanabe, Masashi; Esashi, Masayoshi

The prototype of a solid propellant rocket array thruster for simple attitude control of a 10 kg class micro-spacecraft was completed and tested. The prototype has 10×10 φ0.8 mm solid propellant micro-rockets arrayed at a pitch of 1.2 mm on a 20×22 mm substrate. To realize such a dense array of micro-rockets, each ignition heater is powered from the backside of the thruster through an electrical feedthrough which passes along a propellant cylinder wall. Boron/potassium nitrate propellant (NAB) is used with/without lead rhodanide/potassium chlorate/nitrocellulose ignition aid (RK). Impulse thrust was measured by a pendulum method in air. Ignition required electric power of at least 3 4 W with RK and 4 6 W without RK. Measured impulse thrusts were from 2×10-5 Ns to 3×10-4 Ns after the calculation of compensation for air dumping.

Improved multiple-shot gun for use as a combustion stability rating device

NASA Technical Reports Server (NTRS)

Sokolowski, D. E.

1973-01-01

A program was conducted to develop and experimentally evaluate an improved version of a modified machine gun for use as a device for rating the relative combustion stability of various rocket combustors. Following the results of a previous study involving a caliber .30 machine gun, a caliber .50 machine gun was modified in order to extend the charge-size range of the device. Nitrocellulose charge sizes ranging from 1.004 to 9.720 grams were fired at rates up to four shots per second. Shock pressures up to 25,512 kN/sq m were measured near the end of a shortened gun barrel. A minimal resistance type of check valve permitted the gun to fire into pressurized regions; back pressures up to 3448 kN/sq m abs were tested. The final modified assembly was evaluated during combustion stability tests on rocket combustors burning a FLOX-methane propellant combination.

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.

Comparison of ZetaPlus 60S and nitrocellulose membrane filters for the simultaneous concentration of F-RNA coliphages, porcine teschovirus and porcine adenovirus from river water.

PubMed

Jones, T H; Muehlhauser, V; Thériault, G

2014-09-01

Increasing attention is being paid to the impact of agricultural activities on water quality to understand the impact on public health. F-RNA coliphages have been proposed as viral indicators of fecal contamination while porcine teschovirus (PTV) and porcine adenovirus (PAdV) are proposed indicators of fecal contamination of swine origin. Viruses and coliphages are present in water in very low concentrations and must be concentrated to permit their detection. There is little information comparing the effectiveness of the methods for concentrating F-RNA coliphages with concentration methods for other viruses and vice versa. The objective of this study was to compare 5 current published methods for recovering F-RNA coliphages, PTV and PAdV from river water samples concentrated by electronegative nitrocellulose membrane filters (methods A and B) or electropositive Zeta Plus 60S filters (methods C-E). Method A is used routinely for the detection of coliphages (Méndez et al., 2004) and method C (Brassard et al., 2005) is the official method in Health Canada's compendium for the detection of viruses in bottled mineral or spring water. When river water was inoculated with stocks of F-RNA MS2, PAdV, and PTV to final concentrations of 1×10(6) PFU/100 mL, 1×10(5) gc/100 mL and 3×10(5) gc/100 mL, respectively, a significantly higher recovery for each virus was consistently obtained for method A with recoveries of 52% for MS2, 95% for PAdV, and 1.5% for PTV. When method A was compared with method C for the detection of F-coliphages, PAdV and PTV in river water samples, viruses were detected with higher frequencies and at higher mean numbers with method A than with method C. With method A, F-coliphages were detected in 11/12 samples (5-154 PFU/100 mL), PTV in 12/12 samples (397-10,951 gc/100 mL), PAdV in 1/12 samples (15 gc/100 mL), and F-RNA GIII in 1/12 samples (750 gc/100 mL) while F-RNA genotypes I, II, and IV were not detected by qRT-PCR. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Horizontal semi-dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes.

PubMed

Himber, J

1993-08-01

A high efficiency transfer of the low density lipoprotein (LDL) receptor proteins from polyacrylamide slab gel onto immobilizing nitrocellulose membranes using the horizontal semi-dry electrophoretic system is described. The transfer of the LDL receptors from solubilized rat liver microsomes was performed between two graphite plate electrodes in a continuous buffer system containing methanol and sodium dodecyl sulfate. The protein transfer was achieved in only 150 min at a constant current of 0.8 mA/cm2 at room temperature with very low Joule heat development. The homogeneous electric field yield between the two electrode plates produced a satisfactory transfer of the LDL-receptor protein band in spite of its high molecular weight, and only few protein traces remained in the polyacrylamide gel after blotting. This improved method allows a rapid and quantitative transfer of the LDL receptors without protein denaturation, since the specific binding activity of the blotted receptor is retained as demonstrated by ligand-blotting and immunoblotting.

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

PubMed

Litman, G W; Berger, L; Jahn, C L

1982-06-11

High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions.

Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

PubMed Central

Litman, G W; Berger, L; Jahn, C L

1982-01-01

High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions. Images PMID:6285298

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress.

PubMed

Miladi, Hanene; Soukri, Abdelaziz; Bakhrouf, Amina; Ammar, Emna

2012-11-01

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and -20 °C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18 kDa, as observed on SDS-PAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at -20 °C.

Feasibility study of palm-based fuels for hybrid rocket motor applications

NASA Astrophysics Data System (ADS)

Tarmizi Ahmad, M.; Abidin, Razali; Taha, A. Latif; Anudip, Amzaryi

2018-02-01

This paper describes the combined analysis done in pure palm-based wax that can be used as solid fuel in a hybrid rocket engine. The measurement of pure palm wax calorific value was performed using a bomb calorimeter. An experimental rocket engine and static test stand facility were established. After initial measurement and calibration, repeated procedures were performed. Instrumentation supplies carried out allow fuel regression rate measurements, oxidizer mass flow rates and stearic acid rocket motors measurements. Similar tests are also carried out with stearate acid (from palm oil by-products) dissolved with nitrocellulose and bee solution. Calculated data and experiments show that rates and regression thrust can be achieved even in pure-tested palm-based wax. Additionally, palm-based wax is mixed with beeswax characterized by higher nominal melting temperatures to increase moisturizing points to higher temperatures without affecting regression rate values. Calorie measurements and ballistic experiments were performed on this new fuel formulation. This new formulation promises driving applications in a wide range of temperatures.

Membrane filtration method for enumeration and isolation of Alicyclobacillus spp. from apple juice.

PubMed

Lee, S-Y; Chang, S-S; Shin, J-H; Kang, D-H

2007-11-01

To evaluate the applicability of filtration membranes for detecting Alicyclobacillus spp. spores in apple juice. Ten types of nitrocellulose membrane filters from five manufacturers were used to collect and enumerate five Alicyclobacillus spore isolates and results were compared to conventional K agar plating. Spore recovery differed among filters with an average recovery rate of 126.2%. Recovery levels also differed among spore isolates. Although significant difference (P < 0.05) in spore sizes existed, no correlation could be determined between spore size and membrane filter recovery rate. Recovery of spores using membrane filtration is dependent on the manufacturer and filter pore size. Correlations between spore recovery rate and spore size could not be determined. Low numbers of Alicyclobacillus spores in juice can be effectively detected using membrane filtration although recovery rate differences exist among different manufacturers. Use of membrane filtration is a simple, fast alternative to the week-long enrichment procedures currently employed in most quality assurance tests.

Killing of Listeria monocytogens by conventional and germfree rat sera.

PubMed Central

Czuprynski, C J; Balish, E

1981-01-01

Serum from both germfree and conventional rats, but not plasma or plasma serum, killed Listeria monocytogenes in vitro by a calcium-dependent mechanism that was independent of either complement or lysozyme and was not inhibited by the addition of iron. The listericidin was purified by passing either rat serum or platelet lysate through a nitrocellulose filter (0.2 micrometer) and eluting the activity from the filter with 0.02 N HCl. The partially purified listericidin was heat stable (56 degrees C for 30 min), removed by absorption with zymosan or bentonite, sensitive to treatment with trypsin or pronase, and inhibited by the addition of citrate (0.045 M), suggesting that the serum listericidin is a cationic protein. The development of serum listericidal activity, which could be important in the innate resistance of rats to L. monocytogenes, was dependent on both age and microbial status. Although some discrepancies exist between the serum listericidin and previous descriptions of serum beta-lysin, we believe that the rat serum listericidin is a similar cationic protein. PMID:6792076

Reverse phase protein microarrays: fluorometric and colorimetric detection.

PubMed

Gallagher, Rosa I; Silvestri, Alessandra; Petricoin, Emanuel F; Liotta, Lance A; Espina, Virginia

2011-01-01

The Reverse Phase Protein Microarray (RPMA) is an array platform used to quantitate proteins and their posttranslationally modified forms. RPMAs are applicable for profiling key cellular signaling pathways and protein networks, allowing direct comparison of the activation state of proteins from multiple samples within the same array. The RPMA format consists of proteins immobilized directly on a nitrocellulose substratum. The analyte is subsequently probed with a primary antibody and a series of reagents for signal amplification and detection. Due to the diversity, low concentration, and large dynamic range of protein analytes, RPMAs require stringent signal amplification methods, high quality image acquisition, and software capable of precisely analyzing spot intensities on an array. Microarray detection strategies can be either fluorescent or colorimetric. The choice of a detection system depends on (a) the expected analyte concentration, (b) type of microarray imaging system, and (c) type of sample. The focus of this chapter is to describe RPMA detection and imaging using fluorescent and colorimetric (diaminobenzidine (DAB)) methods.

Analysis of solid propellant combustion in a closed vessel including secondary reaction

NASA Technical Reports Server (NTRS)

Benreuven, M.; Summerfield, M.

1980-01-01

A theory for combustion of solid propellants in a closed vessel is presented allowing for residual exothermic chemical reaction in the bulk of the gas in the vessel. Particular attention is given to propellants exhibiting thick gaseous flame zones such as nitrocellulose, double-base and nitramine propellants. For these, the reaction at high pressures is assumed to involve mainly the oxidation of residual hydrocarbons by NO. It is shown that the direct dynamic coupling between the exothermicity, the molecular weight reduction and the changing pressure can influence the dp/dt-p traces obtained, in a manner not directly related to mass burning rate of the solid. Energy and species conservation equations are derived for the bulk of the vessel in differential form; the system is solved numerically. The results show the effect of extended chemical reaction upon measurable combustion characteristics such as dp/dt-p and burn rate pressure exponent, demonstrating its potential importance in interpretation of closed vessel firing data, depending on the pace of the residual gas phase reactions.

Cell-phone-based measurement of TSH using Mie scatter optimized lateral flow assays.

PubMed

You, David J; Park, Tu San; Yoon, Jeong-Yeol

2013-02-15

Semi-quantitative thyr oid stimulating hormone (TSH) lateral flow immunochromatographic assays (LFA) are used to screen for serum TSH concentration >5 mIUL(-1) (hypothyroidism). The LFA format, however, is unable to measure TSH in the normal range or detect suppressed levels of TSH (<0.4 mIU L(-1); hyperthyroidism). In fact, it does not provide quantitative TSH values at all. Obtaining quantitative TSH results, especially in the low concentration range, has until now required the use of centralized clinical laboratories which require specimen transport, specialized equipment and personnel, and result in increased cost and delays in the timely reporting of important clinical results. We have conducted a series of experiments to develop and validate an optical system and image analysis algorithm based upon a cell phone platform. It is able to provide point-of-care quantitative TSH results with a high level of sensitivity and reproducibility comparable to that of a clinical laboratory-based third-generation TSH immunoassay. Our research approach uses the methodology of the optimized Rayleigh/Mie scatter detection by taking into consideration the optical characteristics of a nitrocellulose membrane and gold nanoparticles on an LFA for quantifying TSH levels. Using a miniature spectrometer, LED light source, and optical fibers on a rotating benchtop apparatus, the light intensity from different angles of incident light and angles of detection to the LFA were measured. The optimum angles were found that the minimized Mie scattering from nitrocellulose membrane, consequently maximizes the Rayleigh scatter detection from the gold nanoparticles in the LFA bands. Using the results from the benchtop apparatus, a cell-phone-based apparatus was designed which utilized the embedded flash in the cell phone camera as the light source, piped the light with an optical fiber from the flash through a collimating lens to illuminate the LFA. Quantification of TSH was performed in an iOS application directly on the phone and verified using the code written in MATLAB. The limit of detection of the system was determined to be 0.31 mIU L(-1) (never achieved before on an LFA format), below the commonly accepted minimum concentration of 0.4 mIU L(-1) indicating clinical significance of hyperthyroidism. The system was further evaluated using human serum showing an accurate and reproducible platform for rapid and point-of-care quantification of TSH using a cell phone. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

PubMed

Dinca, V; Ranella, A; Farsari, M; Kafetzopoulos, D; Dinescu, M; Popescu, A; Fotakis, C

2008-10-01

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Spatial and temporal control of microwave triggered chemiluminescence: a protein detection platform.

PubMed

Previte, Michael J R; Aslan, Kadir; Geddes, Chris D

2007-09-15

We have combined the principles of microwave circuitry and antenna design and our recent work in microwave-triggered metal-enhanced chemiluminescence to now "trigger" chemically and enzyme-catalyzed chemiluminescent reactions with spatial and temporal control. With this technology platform, we achieve spatial and temporal control of enzyme and chemically catalyzed chemiluminescence reactions to achieve more than 500-fold increases in "on-demand" photon flux from chemically catalyzed chemiluminescent reactions. We also report a 6-fold increase in photon flux from HRP-catalyzed assays on disposable coverslips functionalized with HRP and placed proximal to the substrates modified with thin-film aluminum triangle disjointed "bow-tie" structures. In addition, we demonstrate the applicability of this technology to develop multiplexed or high-throughput chemiluminescent assays. We also demonstrate the clinical and biological relevance of this technology platform by affixing aluminum structures in proximity to HRP protein immobilized on nitrocellulose to improve the sensitivity for this model Western blot scheme by 50-fold. We believe analytical applications that rely on enzyme-catalyzed chemiluminescence, such as immunoassays, may greatly benefit from this new platform technology.

A Dual Electrochemical Sensor Based on a Test-strip Assay for the Quantitative Determination of Albumin and Creatinine.

PubMed

Yasukawa, Tomoyuki; Kiba, Yuya; Mizutani, Fumio

2015-01-01

A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6(3-) to Fe(CN)6(4-), which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL(-1), respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

Preparing and Study the effects of Composite Coatings in Protection of Oil Pipes from the Risk of Corrosion that resulting from Associated water with Petroleum Products

NASA Astrophysics Data System (ADS)

– Sarraf, A. R. Al; Yaseen, M. A.

2018-05-01

In order to inhibit the metallic corrosion in the oil pipelines,the protection method with composite coating of unsaturated polyester and reinforced by Caolin at weight percentage (20%) was studied. Where, the work samples were classified into two groups according to internal composite coatings layers for all groups of these samples. The first group is nitrocellulose coating reinforced by nano and micro powder of Mgo, the second group is sodium silicate coating reinforced by nano powder of Mgo. The following weight percentages (0%, 1%, 3% and 5%) were adopted as reinforcement ratios for nano powders, as well as the weight percentages (0%, 3%, 5% and 7%) as reinforcement ratios for micro powders Tribology properties and Electrochemical Corrosion Resistance by Polarization method (Tafel) and Adhesion Strength were studied. The results showed an improvement in the corrosion resistance of protected steel by coatings compare with uncoated steel, as well as improvement in mechanical properties and adhesion strength of composite coatings.

Development of a one-step immunochromatographic strip test for the detection of sennosides A and B.

PubMed

Putalun, Waraporn; Morinaga, Osamu; Tanaka, Hiroyuki; Shoyama, Yukihiro

2004-01-01

An immunochromatographic strip test was developed to detect sennoside A (1) and sennoside B (2) using anti-1 and anti-2 monoclonal antibodies. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of colloidal gold particles coated with the respective sennoside antibodies. The capture reagents were 1- and 2-human serum albumin (HSA) conjugates immobilised on a nitrocellulose membrane on the test strip. The sample containing 1 and 2, together with detector reagent, passed over the zone where the capture reagents had been immobilised. The analytes in the sample competed for binding to the limited amount of antibodies in the detector reagent with the immobilised 1- and 2-HSA conjugates on the membrane and hence positive samples showed no colour in the capture spot zone. Detection limits for the strip test were 125 ng/mL for both sennosides. The assay system is useful as a rapid and simple screening method for the detection of 1 and 2 in plants, drugs and body fluids.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

NASA Technical Reports Server (NTRS)

Perdue, D. O.; Lomax, T. L.

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Multi-immunosensors based on electrolite-insulator-semiconductor structures for determination of some herbicides

NASA Astrophysics Data System (ADS)

Starodub, Nickolaj F.; Starodub, Valentyna M.; Krivenchuk, Vladimir E.; Shapovalenko, Valentyna F.

2002-02-01

New type of the multi-immune sensor was elaborated. It is based on electrolyte-insulator-semiconductors structures and intended for determination of such herbicides as simazine, atrazine and 2,4-D. The specific antibodies were immobilized on nitrocellulose disks, which were placed in measuring cells. The analysis was fulfilled by sequential saturation of antibodies, left unbound after their exposure to native herbicide in investigated sample, with labelled herbicide. If horse radish peroxidase (HRP) was used as label the sensitivity of this multi-immune sensor was about 5 and 1.25 (mu) g/L for simazine and 2,4-D, respectively. At the changing of HRP by (beta) -glucose oxidase the sensitivity of analysis of these herbicides increased approximately in 5 times. The linear plots of the registered concentrations were in the range of 1,0-150,0 and 0,25-150,0 ng/mL for simazine and 2,4-D respectively. It was recommended to use the developed immune sensor for wide screening of herbicides in environment. The ways for increasing of its sensitivity were proposed.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

PubMed

Perdue, D O; Lomax, T L

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Towards an Integrated QR Code Biosensor: Light-Driven Sample Acquisition and Bacterial Cellulose Paper Substrate.

PubMed

Yuan, Mingquan; Jiang, Qisheng; Liu, Keng-Ku; Singamaneni, Srikanth; Chakrabartty, Shantanu

2018-06-01

This paper addresses two key challenges toward an integrated forward error-correcting biosensor based on our previously reported self-assembled quick-response (QR) code. The first challenge involves the choice of the paper substrate for printing and self-assembling the QR code. We have compared four different substrates that includes regular printing paper, Whatman filter paper, nitrocellulose membrane and lab synthesized bacterial cellulose. We report that out of the four substrates bacterial cellulose outperforms the others in terms of probe (gold nanorods) and ink retention capability. The second challenge involves remote activation of the analyte sampling and the QR code self-assembly process. In this paper, we use light as a trigger signal and a graphite layer as a light-absorbing material. The resulting change in temperature due to infrared absorption leads to a temperature gradient that then exerts a diffusive force driving the analyte toward the regions of self-assembly. The working principle has been verified in this paper using assembled biosensor prototypes where we demonstrate higher sample flow rate due to light induced thermal gradients.

On the combustion mechanisms of ZrH2 in double-base propellant.

PubMed

Yang, Yanjing; Zhao, Fengqi; Yuan, Zhifeng; Wang, Ying; An, Ting; Chen, Xueli; Xuan, Chunlei; Zhang, Jiankan

2017-12-13

Metal hydrides are regarded as a series of promising hydrogen-supplying fuel for solid rocket propellants. Their effects on the energetic and combustion performances of propellants are closely related to their reaction mechanisms. Here we report a first attempt to determine the reaction mechanism of ZrH 2 , a high-density metal hydride, in the combustion of a double-base propellant to evaluate its potential as a fuel. ZrH 2 is determined to possess good resistance to oxidation by nitrocellulose and nitroglycerine. Thus its combustion starts with dehydrogenation to generate H 2 and metallic Zr. Subsequently, the newly formed Zr and H 2 participate in the combustion and, especially, Zr melts and then combusts on the burning surface which favors the heat feedback to the propellant. This phenomenon is completely different from the combustion behavior of the traditional fuel Al, where the Al particles are ejected off the burning surface of the propellant to get into the luminous flame zone to burn. The findings in this work validate the potential of ZrH 2 as a hydrogen-supplying fuel for double-base propellants.

Development of a new sensitive immunostrip assay based on mesoporous silica and colloidal Au nanoparticles.

PubMed

Omidfar, Kobra; Khorsand, Behnosh; Larijani, Bagher

2012-02-01

A new competitive immunostrip assay was developed to detect human serum albumin (HSA) in urine sample with use of conjugated monoclonal antibody gold nanoparticles (mAb-AuNPs) and mobile crystalline material (MCM)-41-HSA bioconjugate. To prepare the immunostrip, the colloidal AuNPs with an average particle diameter of 20 nm, was synthesized, labeled with antibody and applied on the conjugate pad as the detection reagent. Then, HSA was attached to the MCM-41 mesoporous nanoparticles and immobilized to a nitrocellulose membrane as the test line. In the optimized investigational conditions, the immunostrip could detect HSA in a high linear range (from 1 to 200 μg/ml) and low detection limit (ng/ml). The reliability of the testing procedure was examined by performing the immunostrip test with 30 urine samples and comparing the results with those obtained via immunoturbidimetry. The immunostrip was adequately sensitive and accurate for a rapid screening of HSA in the urine. This new strategy for competitive immunostrip design can be used and developed for other antigen based immunostrip assay.

Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

PubMed

Johnson, G G; Geiduschek, E P

1977-04-05

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.

Anisotropic membranes for gas separation

DOEpatents

Gollan, A.Z.

1987-07-21

A gas separation membrane has a dense separating layer about 10,000 Angstroms or less thick and a porous support layer 10 to 400 microns thick that is an integral unit with gradually and continuously decreasing pore size from the base of the support layer to the surface of the thin separating layer and is made from a casting solution comprising ethyl cellulose and ethyl cellulose-based blends, typically greater than 47.5 ethoxyl content ethyl cellulose blended with compatible second polymers, such as nitrocellulose. The polymer content of the casting solution is from about 10% to about 35% by weight of the total solution with up to about 50% of this polymer weight a compatible second polymer to the ethyl cellulose in a volatile solvent such as isopropanol, methylacetate, methanol, ethanol, and acetone. Typical nonsolvents for the casting solutions include water and formamide. The casting solution is cast in air from about zero to 10 seconds to allow the volatile solvent to evaporate and then quenched in a coagulation bath, typically water, at a temperature of 7--25 C and then air dried at ambient temperature, typically 10--30 C. 2 figs.

[Starting with camphor--the progress of Nippon Fine Chemical].

PubMed

Kimura, Osamu

2010-01-01

In 1918, Nippon Fine Chemical Co., Ltd. (NFC) was founded under the name, Nippon Camphor Co., Ltd. for the purpose of unifying the camphor business throughout Japan. The company manufactured purified camphor as a government-monopolized good. Camphor was used as a plasticizer for nitrocellulose, as a moth repellent, as an antimicrobial substance, as a rust inhibitor, and as an active ingredient in medicine. It was also a very important good exported in order to obtain foreign currency. Later on, after World War II and the abolition of the camphor monopoly, the company started manufacturing products related to oils and fats, including higher fatty acids, and expanded its business by developing a new field of chemical industry. In 1971 the company changed its name to Nippon Fine Chemical Co., Ltd., and made a new start as a diversified fine chemicals company. Recently, the fine chemicals division of NFC has concentrated on rather complex molecules, such as active pharmaceutical ingredients, and other chemicals. Since 2000, NFC have started to supply "Presome", precursors of liposome DDS drugs. NFC is strengthening marketing strategies in foreign countries with unique technologies and products.

Detection of antibodies to egg drop syndrome virus in chicken serum using a field-based immunofiltration (flow-through) test.

PubMed

Raj, G Dhinakar; Thiagarajan, V; Nachimuthu, K

2007-09-01

A simple, user-friendly, and rapid method to detect the presence of antibodies to egg drop syndrome 76 (EDS) virus in chicken sera based on an immunofiltration (flow-through) test was developed. Purified EDS virus antigen was coated onto nitrocellulose membranes housed in a plastic module with layers of absorbent filter pads underneath. Following addition of serum to be tested and washing, monoclonal antibodies or polyclonal serum to chicken immunoglobulin G (IgG) was used as a bridge antibody to mediate binding between EDS virus-specific IgG and protein A gold conjugate. The appearance of a pink dot indicated the presence of antibodies to EDS virus in the sample tested. The results could be obtained within 5-10 min. The developed immunofiltration test could detect antibodies in the sera of experimentally vaccinated chickens from 2 wk postvaccination. With field sera samples, this test was positive in samples having hemagglutination inhibition titers of 8 and above. This test has the potential to be used as a field-based kit to assess seroconversion in EDS-vaccinated flocks.

Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

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

Honke, K.; Taniguchi, N.; Makita, A.

1986-01-01

A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reactionmore » product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6 ..mu..M, respectively.« less

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Influence of membrane fouling reducers (MFRs) on filterability of disperse mixed liquor of jet loop bioreactors.

PubMed

Koseoglu-Imer, Derya Yuksel; Dizge, Nadir; Karagunduz, Ahmet; Keskinler, Bulent

2011-07-01

The effects of membrane fouling reducers (MFRs) (the cationic polyelectrolyte (CPE) and FeCI(3)) on membrane fouling were studied in a lab-scale jet loop submerged membrane bioreactor (JL-SMBR) system. The optimum dosages of MFRs (CPE dosage=20 mg g(-1)MLSS, FeCI(3) dosage=14 mg g(-1)MLSS) were continuously fed to JL-SMBR system. The soluble and bound EPS concentrations as well as MLSS concentration in the mixed liquor of JL-SMBR were not changed substantially by the addition of MFRs. However, significant differences were observed in particle size and relative hydrophobicity. Filtration tests were performed by using different membrane types (polycarbonate (PC) and nitrocellulose mixed ester (ME)) and various pore sizes (0.45-0.22-0.1 μm). The steady state fluxes (J(ss)) of membranes increased at all membranes after MFRs addition to JL-SMBR. The filtration results showed that MFRs addition was an effective approach in terms of improvement in filtration performance for both membrane types. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field

PubMed Central

Adil, B; Naveen Kumar, B. T.; Patil, Rajreddy; Ballyaya, Abhiman; Ramesh, K. S.; Poojary, Sathish Rama; Byadgi, Omkar V.; Siriyappagouder, Prabhugouda

2013-01-01

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 µg/mL for A. invadans compared to 56 µg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10-11 dilution compared to a 10-8 dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8℃. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish. PMID:23820211

Alteration of blue pigment in artificial iris in ocular prosthesis: effect of paint, drying method and artificial aging.

PubMed

Goiato, Marcelo Coelho; Fernandes, Aline Úrsula Rocha; dos Santos, Daniela Micheline; Hadadd, Marcela Filié; Moreno, Amália; Pesqueira, Aldiéris Alves

2011-02-01

The artificial iris is the structure responsible for the dissimulation and aesthetics of ocular prosthesis. The objective of the present study was to evaluate the color stability of artificial iris of microwaveable polymerized ocular prosthesis, as a function of paint type, drying method and accelerated aging. A total of 40 discs of microwaveable polymerized acrylic resin were fabricated, and divided according to the blue paint type (n = 5): hydrosoluble acrylic, nitrocellulose automotive, hydrosoluble gouache and oil paints. Paints where dried either at natural or at infrared light bulb method. Each specimen was constituted of one disc in colorless acrylic resin and another colored with a basic sclera pigment. Painting was performed in one surface of one of the discs. The specimens were submitted to an artificial aging chamber under ultraviolet light, during 1008 h. A reflective spectrophotometer was used to evaluate color changes. Data were evaluated by 3-way repeated-measures ANOVA and the Tukey HSD test (α = 0.05). All paints suffered color alteration. The oil paint presented the highest color resistance to artificial aging regardless of drying method. Copyright © 2010 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Ultrasonic-based membrane aided sample preparation of urine proteomes.

PubMed

Jesus, Jemmyson Romário; Santos, Hugo M; López-Fernández, H; Lodeiro, Carlos; Arruda, Marco Aurélio Zezzi; Capelo, J L

2018-02-01

A new ultrafast ultrasonic-based method for shotgun proteomics as well as label-free protein quantification in urine samples is developed. The method first separates the urine proteins using nitrocellulose-based membranes and then proteins are in-membrane digested using trypsin. The enzymatic digestion process is accelerated from overnight to four minutes using a sonoreactor ultrasonic device. Overall, the sample treatment pipeline comprising protein separation, digestion and identification is done in just 3h. The process is assessed using urine of healthy volunteers. The method shows that male can be differentiated from female using the protein content of urine in a fast, easy and straightforward way. 232 and 226 proteins are identified in urine of male and female, respectively. From this, 162 are common to both genders, whilst 70 are unique to male and 64 to female. From the 162 common proteins, 13 are present at levels statistically different (p < 0.05). The method matches the analytical minimalism concept as outlined by Halls, as each stage of this analysis is evaluated to minimize the time, cost, sample requirement, reagent consumption, energy requirements and production of waste products. Copyright © 2017 Elsevier B.V. All rights reserved.

Application and evaluation of enzyme-linked immunosorbent assay and immunoblotting for detection of antibodies to Treponema hyodysenteriae in swine.

PubMed Central

Smith, S. C.; Barrett, L. M.; Muir, T.; Christopher, W. L.; Coloe, P. J.

1991-01-01

An enzyme-linked immunoassay (ELISA) has been developed to detect serum Immunoglobulin antibodies G and M to Treponema hyodysenteriae in vaccinated, experimentally infected and naturally infected swine. Naturally infected swine gave ELISA titres that were similar to experimentally infected swine, but were significantly less than the titres of vaccinated swine. When serum from naturally infected swine was used to probe nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresed whole cell proteins of T. hyodysenteriae, the immunoblotting patterns showed IgG antibodies were produced against many T. hyodysenteriae protein antigens and against lipopolysaccharide (LPS). The IgG antibodies directed against LPS were serotype-specific for that LPS and could be used to identify the serotype involved in the T. hyodysenteriae infection in that herd. IgM immunoblots also reacted with the many protein antigens but were less specific for LPS antigen, with a substantial degree of cross-reaction between the LPS of all serotypes. The data demonstrate that a microplate enzyme-linked immunosorbent assay, coupled with immunoblotting, is a very specific and sensitive test for detection of antibody to Treponema hyodysenteriae in swine. Images Fig. 3 Fig. 4 PMID:1936151

Immunoglobulin G responses to Malassezia pachydermatis in healthy dogs and dogs with Malassezia dermatitis.

PubMed

Bond, R; Lloyd, D H

2002-04-20

Serum immunoglobulin G (IgG) responses of healthy dogs and dogs with Malasseziapachydermatis dermatitis were compared by Western immunoblotting. M pachydermatis CBS 1879 was disrupted mechanically and its proteins were separated and blotted on to nitrocellulose membranes before being incubated with sera from eight healthy beagles, eight Irish setters with gluten-sensitive enteropathy, 15 healthy basset hounds, and 30 dogs with Mpachydermatis-associated dermatitis, 20 of which were basset hounds. The mean (se) numbers of bands of immunoreactivity observed in the seborrhoeic basset hounds (10.7 [0.4]) and affected mixed-breed dogs (9.4 [0.9]) were significantly greater than in the beagles (3-0 [1.0]), Irish setters (5.5 [1.1]) and healthy basset hounds (5.6 [0.7]). The number of bands identified was correlated (r(s) = 0.76, P < 0.001) with the anti-M pachydermatis IgG values measured by ELISA in a previous study. Most of the dogs were immunoreactive towards the 132, 66 and 50 to 54 kDa proteins and the affected dogs were also usually reactive towards the 219, 110, 71 and 42 kDa proteins.

Anisotropic membranes for gas separation

DOEpatents

Gollan, Arye Z.

1987-01-01

A gas separation membrane has a dense separating layer about 10,000 Angstroms or less thick and a porous support layer 10 to 400 microns thick that is an integral unit with gradually and continuously decreasing pore size from the base of the support layer to the surface of the thin separating layer and is made from a casting solution comprising ethyl cellulose and ethyl cellulose-based blends, typically greater than 47.5 ethoxyl content ethyl cellulose blended with compatible second polymers, such as nitrocellulose. The polymer content of the casting solution is from about 10% to about 35% by weight of the total solution with up to about 50% of this polymer weight a compatible second polymer to the ethyl cellulose in a volatile solvent such as isopropanol, methylacetate, methanol, ethanol, and acetone. Typical nonsolvents for the casting solutions include water and formamide. The casting solution is cast in air from about zero to 10 seconds to allow the volatile solvent to evaporate and then quenched in a coagulation bath, typically water, at a temperature of 7.degree.-25.degree. C. and then air dried at ambient temperature, typically 10.degree.-30.degree. C.

Installation-restoration program environmental technology development. Task Order 12. Field demonstration - composting of propellant-contaminated sediments at the Badger Army Ammunition Plant (BAAP). Final report, Jul 87-Mar 89

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

Williams, R.T.; Ziegenfuss, P.S.; Marks, P.J.

1989-03-01

A field-scale demonstration of composting propellants-contaminated sediment was conducted at the Badger Army Ammunition Plant (BAAP). Composting, as used at BAAP, is a treatment process in which organic-chemical contaminated soils or sediments are mixed with organic materials such as manure to enhance the role of microbial metabolism in degrading and stabilizing soil/sediment contaminants. Sediments contaminated with the propellant nitrocellulose (NC) were mixed with manure, alfalfa, livestock feed, and wood chips and composted in four static piles. Negative pressure aeration was used to maintain aerobiosis and remove excess heat. Experimental variables investigated during the study were temperature (mesophilic, 35 C vs.more » thermophilic, 55 C), sediment loading (19 to 32 weight percent), and NC loading. Small aliquots of compost (approximately 400 cu cm) were spiked with pure NC, placed in porous nylon bags and buried in compost piles. These bagged compost samples were used to determine if high levels of NC could be successfully composted. Thermophilic temperatures resulted in the highest percent reduction in NC concentration.« less

Redefining Molecular Amphipathicity in Reversing the "Coffee-Ring Effect": Implications for Single Base Mutation Detection.

PubMed

Huang, Chi; Wang, Jie; Lv, Xiaobo; Liu, Liu; Liang, Ling; Hu, Wei; Luo, Changliang; Wang, Fubing; Yuan, Quan

2018-05-21

The "coffee ring effect" is a natural phenomenon where sessile drops leave ring-shaped structures on solid surfaces upon drying. It drives non-uniform deposition of suspended compounds on substrates, which adversely affects many processes, including surface-assisted biosensing and molecular self-assembly. In this study, we describe how the coffee ring effect can be eliminated by controlling the amphipathicity of the suspended compounds, for example DNA modified with hydrophobic dye. Specifically, nuclease digestion of the hydrophilic DNA end converts the dye-labeled molecule into an amphipathic molecule (one with comparably weighted hydrophobic and hydrophilic ends) and reverses the coffee ring effect and results in uniform disc-shaped feature deposition of the dye. The amphipathic product decreases the surface tension of the sessile drops and induces Marangoni flow, which drives the uniform distribution of the amphipathic dye-labeled product in the drops. As proof-of-concept, this strategy was used in a novel enzymatic amplification method for biosensing to eliminate the coffee ring effect on a nitrocellulose membrane and increase assay reliability and sensitivity. Importantly, the reported strategy for eliminating the coffee ring effect can be extended to other sessile drop systems for potentially improving assay reliability, and sensitivity.

Propellant's differentiation using FTIR-photoacoustic detection for forensic studies of improvised explosive devices.

PubMed

Álvarez, Ángela; Yáñez, Jorge; Contreras, David; Saavedra, Renato; Sáez, Pedro; Amarasiriwardena, Dulasiri

2017-11-01

The use of propellant for making improvised explosive devices (IED) is an incipient criminal practice. Propellant can be used as initiator in explosive mixtures along with other components such as coal, ammonium nitrate, sulfur, etc. The identification of the propellant's brand used in homemade explosives can provide additional forensic information of this evidence. In this work, four of the most common propellant brands were characterized by Fourier-transform infrared photoacoustic spectroscopy (FTIR-PAS) which is a non-destructive micro-analytical technique. Spectra shows characteristic signals of typical compounds in the propellants, such as nitrocellulose, nitroglycerin, guanidine, diphenylamine, etc. The differentiation of propellant components was achieved by using FTIR-PAS combined with chemometric methods of classification. Principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) were used to achieve an effective differentiation and classification (100%) of propellant brands. Furthermore, propellant brand differentiation was also assessed using partial least squares discriminant analyses (PLS-DA) by leave one out cross (∼97%) and external (∼100%) validation method. Our results show the ability of FTIR-PAS combined with chemometric analysis to identify and differentiate propellant brands in different explosive formulations of IED. Copyright © 2017 Elsevier B.V. All rights reserved.

Self-Powered Wireless Affinity-Based Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled RFID Antennas.

PubMed

Yuan, Mingquan; Alocilja, Evangelyn C; Chakrabartty, Shantanu

2016-08-01

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Stable isotopes of nitrate reflect natural attenuation of propellant residues on military training ranges.

PubMed

Bordeleau, Geneviève; Savard, Martine M; Martel, Richard; Smirnoff, Anna; Ampleman, Guy; Thiboutot, Sonia

2013-08-06

Nitroglycerin (NG) and nitrocellulose (NC) are constituents of double-base propellants used notably for firing antitank ammunitions. Nitroglycerin was detected in soil and water samples from the unsaturated zone (pore water) at an active antitank firing position, where the presence of high nitrate (NO3(-)) concentrations suggests that natural attenuation of NG is occurring. However, concentrations alone cannot assess if NG is the source of NO3(-), nor can they determine which degradation processes are involved. To address this issue, isotopic ratios (δ(15)N, δ(18)O) were measured for NO3(-) produced from NG and NC through various controlled degradation processes and compared with ratios measured in field pore water samples. Results indicate that propellant combustion and degradation mediated by soil organic carbon produced the observed NO3(-) in pore water at this site. Moreover, isotopic results are presented for NO3(-) produced through photolysis of propellant constituents, which could be a dominant process at other sites. The isotopic data presented here constitute novel information regarding a source of NO3(-) that was practically not documented before and a basis to study the contamination by energetic materials in different contexts.

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

PubMed Central

Worobec, E A; Taneja, A K; Hodges, R S; Paranchych, W

1983-01-01

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites. Images PMID:6185467

Integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching "absolute" zero background in immunoassays.

PubMed

Ma, Hongwei; Wu, Yuanzi; Yang, Xiaoli; Liu, Xing; He, Jianan; Fu, Long; Wang, Jie; Xu, Hongke; Shi, Yi; Zhong, Renqian

2010-08-01

The key to achieve a highly sensitive and specific protein microarray assay is to prevent nonspecific protein adsorption to an "absolute" zero level because any signal amplification method will simultaneously amplify signal and noise. Here, we develop a novel solid supporting material, namely, polymer coated initiator integrated poly(dimethysiloxane) (iPDMS), which was able to achieve such "absolute" zero (i.e., below the detection limit of instrument). The implementation of this iPDMS enables practical and high-quality multiplexed enzyme-linked immunosorbent assay (ELISA) of 11 tumor markers. This iPDMS does not need any blocking steps and only require mild washing conditions. It also uses on an average 8-fold less capture antibodies compared with the mainstream nitrocellulose (NC) film. Besides saving time and materials, iPDMS achieved a limit-of-detection (LOD) as low as 19 pg mL(-1), which is sufficiently low for most current clinical diagnostic applications. We expect to see an immediate impact of this iPDMS on the realization of the great potential of protein microarray in research and practical uses such as large scale and high-throughput screening, clinical diagnosis, inspection, and quarantine.

A MODIFIED PROTEIN ASSAY FROM MICROGRAM TO LOW NANOGRAM LEVELS IN DILUTE SAMPLES

PubMed Central

Heda, Ghanshyam D.; Kunwar, Upasana; Heda, Rajiv P.

2013-01-01

In this paper we present a modified and improved protein assay that was previously described as ‘amidoschwarz assay’ by Schaffner and Weissmann (Anal. Biochem. 56, 1973, 502–514). Our improved protein assay is user-friendly and 30 to 40 times more sensitive than the earlier method. The assay was developed into 3 formats (maco, micro, and nanoassay) with TCA as protein precipitating agent; measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots, bound to nitrocellulose membrane with lowest protein measurements to 1 μg and 0.1 μg respectively. The nanoassay on the other hand with combination staining of amido black followed by colloidal gold can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 μg/ml, prior to their biochemical analysis such as in comparative proteomics. PMID:24135655

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

PubMed Central

Dernick, Gregor; Obermüller, Stefan; Mangold, Cyrill; Magg, Christine; Matile, Hugues; Gutmann, Oliver; von der Mark, Elisabeth; Handschin, Corinne; Maugeais, Cyrille; Niesor, Eric J.

2011-01-01

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique. PMID:21971713

Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein.

PubMed

Worobec, E A; Taneja, A K; Hodges, R S; Paranchych, W

1983-02-01

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.

Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein.

PubMed

Govender, Nisha; Wong, Mui-Yun

2017-04-01

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

Paper-based immunosensor with signal amplification by enzyme-labeled anti-p16INK4a multifunctionalized gold nanoparticles for cervical cancer screening.

PubMed

Yokchom, Ruchuon; Laiwejpithaya, Somsak; Maneeprakorn, Weerakanya; Tapaneeyakorn, Satita; Rabablert, Jundee; Dharakul, Tararaj

2018-04-01

The aim of this study was to develop a paper-based immunosensor for cervical cancer screening, with signal amplification by multifunctionalized gold nanoparticles (AuNPs). The AuNPs were functionalized with a highly specific antibody to the p16 INK4a cancer biomarker. The signal was amplified using a combination of the peroxidase activity of horseradish peroxidase (HRP) enzyme-antibody conjugate and the peroxidase-like activity of the AuNPs. The immune complex of p16 INK4a protein and multifunctionalized AuNPs was deposited on the nitrocellulose membrane, and a positive result was generated by catalytic oxidation of peroxidase enzyme substrate 3,3',5,5'-Tetramethylbenzidine (TMB). The entire reaction occurred on the membrane within 30 min. Evaluation in clinical samples revealed 85.2% accuracy with a kappa coefficient of 0.69. This proof of concept study demonstrates the successful development of a highly accurate, paper-based immunosensor that is easy to interpret using the naked eye and that is suitable for cervical cancer screening in low-resource settings. Copyright © 2018 Elsevier Inc. All rights reserved.

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos

PubMed Central

Kim, Young Ah; Lee, Eun-Hye; Kim, Kwang-Ok; Lee, Yong Tae; Hammock, Bruce D.; Lee, Hye-Sung

2014-01-01

An immunochromatographic assay (ICA) based on competitive antigen-coated format using colloidal gold as the label was developed for the detection of the organophosphorus insecticide chlorpyrifos. The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with chlorpyrifos hapten-OVA conjugate (test line) and anti-mouse IgG (control line). Based on the fact that the competition is between the migrating analyte in the sample and the analyte hapten immobilized on the test strip for the binding sites of the antibody-colloidal gold (Ab-CG) conjugate migrating on the test strip, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. This criterion was utilized for the confirmation of appropriateness of a nitrocellulose (NC) membrane for chlorpyrifos ICA. The detection limit of the ICA for chlorpyrifos standard and chlorpyrifos spiked into agricultural samples were 10 and 50 ng mL−1, respectively. The assay time for the ICA test was less than 10 min, suitable for rapid on-site testing of chlorpyrifos. PMID:21504817

Lanthanide-functionalized silver nanoparticles for detection of an anthrax biomarker and test paper fabrication

NASA Astrophysics Data System (ADS)

Tan, Hongliang; Li, Qian; Ma, Chanjiao; Song, Yonghai; Xu, Fugang; Chen, Shouhui; Wang, Li

2014-01-01

It is highly desirable to develop a simple and sensitive analytical method for detection of anthrax biomarker (dipicolinic acid, DPA) because of its dangerous nature. In this work, we developed a fluorescent sensor for DPA detection based on terbium ion-functionalized silver nanoparticles with an average size of 6.7 nm (AgNPs-Tb3+). The fluorescent intensity of Tb-DPA complex on the surface of AgNPs was two times higher than that of Tb-DPA complex alone in a solution phase due to the metal-enhanced fluorescence (MEF) effect of AgNPs. The proposed fluorescent sensor exhibits excellent selectivity and high sensitivity for DPA. Importantly, a test paper for DPA detection was fabricated for the first time by the integration of AgNPs-Tb3+ onto the nitrocellulose membrane. Owing to the MEF effect of AgNPs, the lowest detectable concentration of the test paper-integrated AgNPs-Tb3+ for DPA by naked eyes is 10 times lower than that of the test paper-integrated Tb3+ alone. We believe that the presented strategy may open up new avenues to the development of portable and robust-sensing platforms based on functional hybrid materials.

A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams

NASA Astrophysics Data System (ADS)

Chen, Yanni; Wang, Yongwei; Liu, Liqiang; Wu, Xiaoling; Xu, Liguang; Kuang, Hua; Li, Aike; Xu, Chuanlai

2015-10-01

A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples.A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04987c

α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

PubMed Central

Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

1981-01-01

[3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

Badger Army Ammunition Plant groundwater data management system

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

Hansen, J.P.

1994-12-31

At the Badger Army Ammunition Plant (Badger), there are currently over 200 wells that are monitored on a quarterly basis. Badger has had three active production periods since its construction in 1942. During these periods, various nitrocellulose based propellants were produced including single base artillery propellants were produced including single base artillery propellant, double base rocket propellant and BALL POWDER{reg_sign} propellant. Intermediate materials used in the manufacture of these propellants were also produced, including nitroglycerine, and sulfuric and nitric acids. To meet the challenge of managing the data in-house, a groundwater data management system (GDMS) was developed. Although such systemsmore » are commercially available, they were not able to provide the specific capabilities necessary for data management and reporting at Badger. The GDMS not only provides the routine database capabilities of data sorts and queries, but has provided an automated data reporting system as well. The reporting function alone has significantly reduced the time and efforts that would normally be associated with this task. Since the GDMS was developed at Badger, the program can be continually adapted to site specific needs. Future planned modifications include automated reconciliation, improved transfer of data to graphics software, and statistical analysis and interpretation of the data.« less

Ultrarapid electrophoretic transfer of high and low molecular weight proteins using heat.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

An ultrarapid method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7% (0.75 mm) SDS polyacrylamide gel. For 10 and 12.5% gels (0.75 mm) the corresponding time was 15 min. A complete transfer could be carried out in 20 min for 7, 10, and 12.5% gels (1.5 mm gels). The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. The heat mediated transfer method was compared with a conventional transfer protocol, under similar conditions. The conventional method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is particularly useful for the transfer of high molecular weight proteins, very rapid, and avoids the use of methanol.

Western blotting of high and low molecular weight proteins using heat.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

A method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 °C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7 % (0.75 mm) SDS polyacrylamide gel. For 10 and 12.5 % gels (0.75 mm) the corresponding time was 15 min. A complete transfer could be carried out in 20 min for 7, 10, and 12.5 % gels (1.5 mm gels). The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. The heat mediated transfer method was compared with a conventional transfer protocol, under similar conditions. The conventional method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is particularly useful for the transfer of high molecular weight proteins, very rapid, and avoids the use of methanol.

Thrombophilic gene polymorphisms and recurrent pregnancy loss in Greek women.

PubMed

Chatzidimitriou, M; Chatzidimitriou, D; Mavridou, M; Anetakis, C; Chatzopoulou, F; Lialiaris, T; Mitka, S

2017-12-01

Recurrent pregnancy loss (RPL) is a multifactorial disorder. The aim of this study was the detection of various genetic polymorphisms and their correlation to RPL, in Greek women. The impact of 12 thrombophilic polymorphisms was evaluated, among 48 Greek women with a history of RPL, vs 27 healthy parous women. Multiplex PCR and in situ hybridization on nitrocellulose films were performed, to investigate 12 genetic polymorphisms previously reported as risk factors for RPL. Heterozygous FV Leiden, homozygous PAI-1 4G/4G, heterozygous MTHFR C677T, homozygous MTHFR A1298C, as much as the combined thrombophilic genotypes MTHFR 677T + ACE Ι/D, MTHFR 677T/1298C + ACE D/D, ACE I/D + b-fibrinogen -455 G/A, FV HR2 + b-fibrinogen -455 G/A showed a correlation as risk factors for RPL, whereas the rest of the investigated polymorphisms and their combinations did not render statistically significant differences between the two groups in study. The results of this study, as well as those of similar studies, concerning the detection of genetic, environmental, and physiological factors underlying RPL, will prove of critical significance in the investigation and treatment of thrombophilic predisposition, in cases of RPL. © 2017 John Wiley & Sons Ltd.

Resolution of plasma sample mix-ups through comparison of patient antibody patterns to E. coli.

PubMed

Vetter, Beatrice N; Orlowski, Vanessa; Schüpbach, Jörg; Böni, Jürg; Rühe, Bettina; Huder, Jon B

2015-12-01

Accidental sample mix-ups and the need for their swift resolution is a challenge faced by every analytical laboratory. To this end, we developed a simple immunoblot-based method, making use of a patient's characteristic plasma antibody profile to Escherichia coli (E. coli) proteins. Nitrocellulose strips of size-separated proteins from E. coli whole-cell lysates were incubated with patient plasma and visualised with an enzyme-coupled secondary antibody and substrate. Plasma samples of 20 random patients as well as five longitudinal samples of three patients were analysed for antibody band patterns, to evaluate uniqueness and consistency over time, respectively. For sample mix-ups, antibody band patterns of questionable samples were compared with samples of known identity. Comparison of anti-E. coli antibody patterns of 20 random patients showed a unique antibody profile for each patient. Antibody profiles remained consistent over time, as shown for three patients over several years. Three example cases demonstrate the use of this methodology in mis-labelling or -pipetting incidences. Our simple method for resolving plasma sample mix-ups between non-related individuals can be performed with basic laboratory equipment and thus can easily be adopted by analytical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

Nanoporous membranes enable concentration and transport in fully wet paper-based assays.

PubMed

Gong, Max M; Zhang, Pei; MacDonald, Brendan D; Sinton, David

2014-08-19

Low-cost paper-based assays are emerging as the platform for diagnostics worldwide. Paper does not, however, readily enable advanced functionality required for complex diagnostics, such as analyte concentration and controlled analyte transport. That is, after the initial wetting, no further analyte manipulation is possible. Here, we demonstrate active concentration and transport of analytes in fully wet paper-based assays by leveraging nanoporous material (mean pore diameter ≈ 4 nm) and ion concentration polarization. Two classes of devices are developed, an external stamp-like device with the nanoporous material separate from the paper-based assay, and an in-paper device patterned with the nanoporous material. Experimental results demonstrate up to 40-fold concentration of a fluorescent tracer in fully wet paper, and directional transport of the tracer over centimeters with efficiencies up to 96%. In-paper devices are applied to concentrate protein and colored dye, extending their limits of detection from ∼10 to ∼2 pmol/mL and from ∼40 to ∼10 μM, respectively. This approach is demonstrated in nitrocellulose membrane as well as paper, and the added cost of the nanoporous material is very low at ∼0.015 USD per device. The result is a major advance in analyte concentration and manipulation for the growing field of low-cost paper-based assays.

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.

RNA binding and replication by the poliovirus RNA polymerase

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

Oberste, M.S.

1988-01-01

RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs wasmore » examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.« less

Graphene Inks with Cellulosic Dispersants: Development and Applications for Printed Electronics

NASA Astrophysics Data System (ADS)

Secor, Ethan Benjamin

Graphene offers promising opportunities for applications in printed and flexible electronic devices due to its high electrical and thermal conductivity, mechanical flexibility and strength, and chemical and environmental stability. However, scalable production and processing of graphene presents a critical technological challenge preventing the application of graphene for flexible electronic interconnects, electrochemical energy storage, and chemically robust electrical contacts. In this thesis, a promising and versatile platform for the production, patterning, and application of graphene inks is presented based on cellulosic dispersants. Graphene is produced from flake graphite using scalable liquid-phase exfoliation methods, using the polymers ethyl cellulose and nitrocellulose as multifunctional dispersing agents. These cellulose derivatives offer high colloidal stability and broadly tunable rheology for graphene dispersions, providing an effective and tunable platform for graphene ink development. Thermal or photonic annealing decomposes the polymer dispersant to yield high conductivity, flexible graphene patterns for various electronics applications. In particular, the chemical stability of graphene enables robust electrical contacts for ceramic, metallic, organic and electrolytic materials, validating the diverse applicability of graphene in printed electronics. Overall, the strategy for graphene ink design presented here offers a simple, efficient, and versatile method for integrating graphene in a wide range of printed devices and systems, providing both fundamental insight for nanomaterial ink development and realistic opportunities for practical applications.

Expression of the 68-kilodalton neurofilament gene in aluminum intoxication

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

Muma, N.A.; Troncoso, J.C.; Hoffman, P.N.

1986-03-01

Intrathecal administration of aluminum salts induces accumulation of neurofilaments (NFs) in cell bodies and proximal axons of rabbit spinal motor neurons. Mechanisms leading to this pathological change are not well understood. Although impairments of NF transport have been demonstrated in this model, the hypothesis that NF accumulations are the result of an increase in NF synthesis needs to be explored. In rabbits, a large percentage of neurons develop accumulations of NFs following injections of aluminum lactate directly into the cisterna magna or into a reservoir placed in the lateral ventricle. To study levels of mRNA encoding cytoskeletal proteins, spinal cordmore » RNA was extracted, separated on a denaturing agarose gel, transferred to nitrocellulose paper, and hybridized to (/sup 32/P)-labeled cDNA clones encoding the mouse 68-kilodalton (kd) NF subunit and tubulin. Examining a constant amount of RNA, the radioactivity of labeled mRNA bands for the 68-kd NF subunit and for tubulin was decreased in spinal cords of aluminum-treated rabbits. These preliminary results will be followed up by in situ hybridization to determine levels of mRNA for tubulin and 68-kd NF subunit in affected and in normal spinal neurons. In conclusion, administration of aluminum decreased mRNA for the 608-kd NF protein in spinal neurons.« less

A method for obtaining RNA from Hemileia vastatrix appressoria produced in planta, suitable for transcriptomic analyses.

PubMed

Loureiro, Andreia; Azinheira, Helena Gil; Silva, Maria do Céu; Talhinhas, Pedro

2015-11-01

Appressoria are the first infection structures developed by rust fungi and require specific topographic signals from the host for their differentiation. The ease in obtaining appressoria in vitro for these biotrophic fungi led to studies concerning gene expression and gene discovery at appressorial level, avoiding the need to distinguish plant and fungal transcripts. However, in some pathosystems, it was observed that gene expression in appressoria seems to be influenced by host-derived signals, suggesting that transcriptomic analyses performed from in planta differentiated appressoria would be potentially more informative than those from in vitro differentiated appressoria. Nevertheless analysing appressorial RNA obtained from in planta samples is often hampered by an excessive dilution of fungal RNA within plant RNA, besides uncertainty regarding the fungal or plant origin of RNA from highly conserved genes. To circumvent these difficulties, we have recovered Hemileia vastatrix appressoria from Arabica coffee leaf surface using a film of nitrocellulose dissolved in butyl and ethyl acetates (nail polish), and extracted fungal RNA from the polish peel. RNA thus obtained is of good quality and usable for cDNA synthesis and transcriptomic (quantitative PCR) studies. This method could provide the means to investigate specific host-induced appressoria-related fungal pathogenicity factors. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Lipid A binding proteins in macrophages detected by ligand blotting

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

Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

1987-05-01

Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model.

PubMed

Mi, Shengli; Yang, Xueyi; Zhao, Qingmei; Qu, Lei; Chen, Shuming; M Meek, Keith; Dou, Zhongying

2008-11-01

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.

A novel immunochromatographic electrochemical biosensor for highly sensitive and selective detection of trichloropyridinol, a biomarker of exposure to chlorpyrifos.

PubMed

Wang, Limin; Lu, Donglai; Wang, Jun; Du, Dan; Zou, Zhexiang; Wang, Hua; Smith, Jordan N; Timchalk, Charles; Liu, Fengquan; Lin, Yuehe

2011-02-15

We present a novel portable immunochromatographic electrochemical biosensor (IEB) for simple, rapid, and sensitive biomonitoring of trichloropyridinol (TCP), a metabolite biomarker of exposure to organophosphorus insecticides. Our new approach takes the advantage of immunochromatographic test strip for a rapid competitive immunoreaction and a disposable screen-printed carbon electrode for a rapid and sensitive electrochemical analysis of captured HRP labeling. Several key experimental parameters (e.g. immunoreaction time, the amount of HRP labeled TCP, concentration of the substrate for electrochemical measurements, and the blocking agents for the nitrocellulose membrane) were optimized to achieve a high sensitivity, selectivity and stability. Under optimal conditions, the IEB has demonstrated a wide linear range (0.1-100 ng/ml) with a detection limit as low as 0.1 ng/ml TCP. Furthermore, the IEB has been successfully applied for biomonitoring of TCP in the rat plasma samples with in vivo exposure to organophosphorus insecticides like Chlorpyrifos-oxon (CPF-oxon). The IEB thus opens up new pathways for designing a simple, rapid, clinically accurate, and quantitative tool for TCP detection, as well as holds a great promise for in-field screening of metabolite biomarkers, e.g., TCP, for humans exposed to organophosphorus insecticides. Copyright © 2010 Elsevier B.V. All rights reserved.

Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

PubMed Central

Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A.; Atluri, Vidya L.; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A.; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W. John W.; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H.; Vinetz, Joseph M.; Tsolis, Renée M.; Felgner, Philip L.

2010-01-01

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host. PMID:20454614

Rapid Detection of Chloramphenicol Residues in Aquatic Products Using Colloidal Gold Immunochromatographic Assay

PubMed Central

Zhou, Chennan; Zhang, Xueyin; Huang, Xinxin; Guo, Xishan; Cai, Qiang; Zhu, Songming

2014-01-01

A colloidal gold immunochromatographic assay (GICA) was developed for rapid detection of chloramphenicol (CAP) residues in aquatic products. A nitrocellulose (NC) membrane was used as the carrier, and the polyclonal CAP antibody was used as the marker protein. The average diameter of as-prepared colloidal gold nanoparticles (AuNPs) was about 20 nm. The optimal pH value of colloidal gold solutions and the amount of the antibody of CAP were 8.0 and 7.2 μg/mL, respectively. The CAP antibody was immobilized onto the conjugate pad after purification. The CAP conjugate and goat anti-rabbit IgG (secondary antibody) were coated onto the NC membrane. Next, the non-specific sites were blocked with 1% bovine serum albumin. The minimum detectable concentration of CAP in standard solution is 0.5 ng/mL, with good reproducibility. For the real samples from crucian carps injected with a single-dose of CAP in the dorsal muscles, the minimum detectable concentration of CAP residues was 0.5 μg/kg. The chromatographic analysis time was less than 10 min, and the strip had a long storage lifetime of more than 90 days at different temperatures. The strips provide a means for rapid detection of CAP residues in aquatic products. PMID:25412221

Development of a nanogold-based immunochromatographic assay for detection of morphine in urine using the Amor-HK16 monoclonal antibody.

PubMed

Dehghannezhad, Ardeshir; Paknejad, Maliheh; Rasaee, Mohammad Javad; Omidfar, Kobra; Seyyed Ebrahimi, Shadi Sadat; Ghahremani, Hossein

2012-12-01

A simple, rapid competitive immunochromatography (ICG) strip test was developed to detect morphine in urine samples using a monoclonal antibody produced in-house and conjugated to gold nanoparticles. Hybridoma cells were cultured and the Amor-HK16 monoclonal antibody against morphine was obtained from the supernatant after purification by salting out and passing through a Protein G-Agarose affinity column. Morphine was obtained from morphine sulfate and a C6-hemisuccinate derivative of morphine was prepared, conjugated to bovine serum albumin, and immobilized to a nitrocellulose membrane as the test line. Goat anti-mouse antibody was used as a binder in the control line in the detection zone of the strip. Colloidal gold particles of diameter approximately 20 nm were prepared and conjugated to the monoclonal antibody. The detection limit of the test strip was found to be 2000 ng/mL of morphine in urine samples. Reliability was determined by performing the ICG test on 103 urine samples and comparing the results with those obtained by thin-layer chromatography. The sensitivity of the test was 100%, and the analysis time for the assay was approximately 5 min. The new ICG method was adequately sensitive and accurate for the rapid screening of morphine in urine.

In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

PubMed Central

Koji, Takehiko; Ueda, Kazumitsu; Pastan, Ira; Gottesman, Michael M.; Nakane, Paul K.; Mori, Shigeo

1990-01-01

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. PMID:1977730

Biodegradability of regenerated cellulose films coated with polyurethane/natural polymers interpenetrating polymer networks

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

Zhang, L.; Zhou, J.; Huang, J.

1999-11-01

Interpenetrating polymer network (IPN) coatings synthesized from castor-oil-based polyurethane (PU) with chitosan, nitrocellulose, or elaeostearin were coated on regenerated cellulose (RC) film for curing at 80--100 C for 2--5 min, providing biodegradable, water-resistant cellulose films coded, respectively, as RCCH, RCNC, and RCEs. The coated films were buried in natural soil for decaying and inoculated with a spore suspension of fungi on the agar medium, respectively, to test biodegradability. The viscosity-average molecular weight, M{sub {eta}}, and the weight of the degraded films decreased sharply with the progress of degradation. The degradation half-lifes, t{sub 1/2}, of the films in soil at 30more » C were found to be 19 days for RC, 25 days for RCNC, 32 days for RCCH, and 45 days for the RCEs films. Scanning electron microscopy (SEM) showed that the extent of decay followed in the order RC {gt} RCNC {gt} RCCH {gt} RCEs. SEM, infrared (IR), high-performance liquid chromatography (HPLC), and CO{sub 2} evolution results indicated that the microorganisms directly attacked the water-resistant coating layer and then penetrated into the cellulose to speedily metabolize, while accompanying with producing CO{sub 2}, H{sub 2}O, glucose cleaved from cellulose, and small molecules decomposed from the coatings.« less

High frequency of Helicobacter pylori DNA in drinking water in Kermanshah, Iran, during June-November 2012.

PubMed

Amirhooshang, Alvandi; Ramin, Abiri; Ehsan, Aryan; Mansour, Rezaei; Shahram, Bagherabadi

2014-09-01

To gain a better understanding of transmission and selecting appropriate measures for preventing the spread of Helicobacter pylori, the aim of this study was to investigate the prevalence of H. pylori in drinking water samples in Kermanshah, Iran. Drinking water samples were collected from around Kermanshah and filtered through 0.45 μm nitrocellulose filters. The bacterial sediment was subjected to DNA extraction and polymerase chain reaction (PCR) for H. pylori detection using newly designed primers targeted at the conserved region of the ureC gene. The overall detection rates for H. pylori DNA in the water samples were 56% (66/118) with a frequency of 36% (25/70) in tap water samples and 85% (41/48) in wells. The detection limit was 50 bacteria per liter of filtered water and a pure H. pylori DNA copy number of 6 per reaction. Based on the evidence we may suggest that recontamination occurred and H. pylori entered into the water piping system through cracked or broken pipes or was released from established H. pylori biofilms on pipes. In conclusion, a high prevalence of H. pylori was detected in drinking water samples that strengthens the evidence of H. pylori transmission through drinking water.

Protein microarray analysis reveals BAFF-binding autoantibodies in systemic lupus erythematosus

PubMed Central

Price, Jordan V.; Haddon, David J.; Kemmer, Dodge; Delepine, Guillaume; Mandelbaum, Gil; Jarrell, Justin A.; Gupta, Rohit; Balboni, Imelda; Chakravarty, Eliza F.; Sokolove, Jeremy; Shum, Anthony K.; Anderson, Mark S.; Cheng, Mickie H.; Robinson, William H.; Browne, Sarah K.; Holland, Steven M.; Baechler, Emily C.; Utz, Paul J.

2013-01-01

Autoantibodies against cytokines, chemokines, and growth factors inhibit normal immunity and are implicated in inflammatory autoimmune disease and diseases of immune deficiency. In an effort to evaluate serum from autoimmune and immunodeficient patients for Abs against cytokines, chemokines, and growth factors in a high-throughput and unbiased manner, we constructed a multiplex protein microarray for detection of serum factor–binding Abs and used the microarray to detect autoantibody targets in SLE. We designed a nitrocellulose-surface microarray containing human cytokines, chemokines, and other circulating proteins and demonstrated that the array permitted specific detection of serum factor–binding probes. We used the arrays to detect previously described autoantibodies against cytokines in samples from individuals with autoimmune polyendocrine syndrome type 1 and chronic mycobacterial infection. Serum profiling from individuals with SLE revealed that among several targets, elevated IgG autoantibody reactivity to B cell–activating factor (BAFF) was associated with SLE compared with control samples. BAFF reactivity correlated with the severity of disease-associated features, including IFN-α–driven SLE pathology. Our results showed that serum factor protein microarrays facilitate detection of autoantibody reactivity to serum factors in human samples and that BAFF-reactive autoantibodies may be associated with an elevated inflammatory disease state within the spectrum of SLE. PMID:24270423

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

PubMed

Kurzchalia, T V; Wiedmann, M; Breter, H; Zimmermann, W; Bauschke, E; Rapoport, T A

1988-03-15

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease

PubMed Central

2011-01-01

Background In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. Results In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10-5 dilution of Asia1/JSL/05 (1 × 107.2TCID50/50 μL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. Conclusion We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields. PMID:21880157

Development of Lateral Flow Immunochromatographic Strips for Micropollutants Screening Using Colorants of Aptamer Functionalized Nanogold Particles Part I: Methodology and Optimization.

PubMed

Zhao, Shuai; Zhang, Shan; Wang, Sai; Liu, Jiahui; Dong, Yiyang

2018-05-03

A methodology of lateral flow immunochromatographic strip based on aptamer was developed for on-site detectionof the small molecule micropollutants. In the present study, we try for the first time to investigate the feasibility of developing a strip assay for the analysis of micropollutants as methodological prototypes by combining the high selectivity and affinity of aptamers with the unique optical properties of nanogolds. This quantitative method was based on the competition for the aptamer between targets and DNA probes. Crucial parameters that might influence the sensitivity, such as the size of nanogolds, amount of aptamer, type and pH of streptavidin, type of nitrocellulose (NC) membrane, blocking procedure, and reading time, were systematically investigated to obtain the optimum assay performance. With the optimized conditions [nanogolds 25 nm, 50 μM aptamer, pH 8 of GSA (a type of streptavidin named "SA Gold," which is a sulfhydrylization streptavidin), Millipore HFC 135 NC membrane, 1% bovine serum albumin as the blocking agent and added in the running buffer and sample pad soakage agents, and 20 min reading time] the aptamer-based lateral flow assay will show a low visual limit of detection and scanning reader LOD. The strip for on-site screening using colorants of aptamer functionalized nanogold particles did not require any complicated equipment and was a potential portable tool for rapid identification of micropollutants.

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

PubMed Central

Tian, Y; Adya, N; Wagner, S; Giam, C Z; Green, M R; Ellington, A D

1995-01-01

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies. PMID:7489503

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

PubMed Central

Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

1994-01-01

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system. PMID:7496936

Ultrasensitive immunochromatographic assay for the simultaneous detection of five chemicals in drinking water.

PubMed

Xing, Changrui; Liu, Liqiang; Song, Shanshan; Feng, Min; Kuang, Hua; Xu, Chuanlai

2015-04-15

In this paper, we describe the development of a multicomponent lateral-flow assay based on an antibody-antigen reaction for the rapid and simultaneous detection of trace contaminants in water, including a heavy metal, algal toxin, antibiotic, hormone, and pesticide. The representative analytes chosen for the study were lead (Pb(II), microcystin-leucine-arginine (MC-LR), chloramphenicol (CAP), testosterone (T), and chlorothalonil (CTN). Five different antigens were immobilized separately in five test lines on a nitrocellulose membrane. The monoclonal antibodies specifically recognized the corresponding antigens, and there was no cross-reactivity between the antibodies in the detection assay. Samples or standards containing the five analytes were preincubated with the freeze-dried colloidal-gold-labeled monoclonal antibody conjugates to improve the sensitivity of the assay. The results were obtained within 20min with a paper-based sensor. The cut-off values for the strip test were 4ng/mL for Pb(II), 1ng/mL for MC-LR, 0.1ng/mL for CAP, 5ng/mL for T, and 5ng/mL for CTN. The assay was evaluated using spiked water samples, and the accuracy and reproducibility of the results were good. In summary, this lateral-flow device provides an effective and rapid method for the onsite detection of multiple contaminants in water samples, with no treatment or devices required. Copyright © 2014 Elsevier B.V. All rights reserved.

Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

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

Wiktorowicz, J.E.; Whitman, J.M.

1986-05-01

Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated severalmore » more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.« less

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

PubMed

Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

1994-03-01

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

Development of an immunochromatographic strip test for rapid detection of citrus yellow vein clearing virus.

PubMed

Bin, Yu; Li, Zhongan; Wu, Jianxiang; Wang, Xuefeng; Zhou, Yan; Li, Taisheng; Yang, Fangyun; Zhou, Changyong; Song, Zhen

2018-02-01

A rapid immunochromatographic strip (ICS) test for detection of citrus yellow vein clearing virus (CYVCV) was developed. The test is based on an antibody sandwich format and uses the monoclonal antibody (MAb) 1E1, which is specific for CYVCV. MAb 1E1 labeled with 30-nm colloidal gold particles was coated on a gold conjugate pad. A secondary goat anti-mouse IgG was coated on the surface of a nitrocellulose filter membrane (NC) as the control (C) line, while 1E1 was coated on the surface of the NC as the test (T) line. The ICS test was evaluated for specificity and sensitivity and then applied for virus detection in field samples. There was no cross-reaction with citrus tristeza virus (CTV), satsuma dwarf virus (SDV), citrus tatter leaf virus (CTLV), citrus exocortis viroid (CEVd), citrus mosaic virus (CiMV), citrus psorosis virus (CPV), citrus ringspot virus (RSV) or 'Candidatus Liberibacter asiaticus' (CLas). The ICS test was still able to detect CYVCV in tissue extracts at a dilution of 1: 320 (w/v), which is as efficient as the dot-ELISA assay. In general, the ICS assay is less expensive, faster and simpler to conduct than conventional CYVCV detection methods, so it may be useful for large-scale detection or monitoring of CYVCV.

Intercomparison between Individual Particles Scavenged as CCN and Those Collected at Ground-based Site on West Coast of Japan During Asian Dust Storm

NASA Astrophysics Data System (ADS)

Ma, C. J.; Tohno, S.; Kasahara, M.; Hayakawa, S.

2002-12-01

Asian dust storm particles can affect precipitation composition because they are either incorporated into cloud via condensation of water vapour (nucleation) or due to the uptake of particles by existing droplets. And subsequently they affect aquatic and terrestrial ecosystems. In order to study the intercorrelation between the chemical natures of both the particles collected at ground-based site and those scavenged as CCN, the intensive field measurement was carried out on west coast of Japan (Yasaka, Tango Peninsula, 35.62°N; 135.07°E) during dense Asian dust storm event on March 22, 2002. Due to the size dependence of the chemical composition of aerosol particle, size-segregated aerosol particles were collect using Low pressure Andersen impactor sampler. Also, to collect cloud droplets individually, a particular method for the cloud droplet replication was newly applied using collodion (nitrocellulose) film. Sampling of cloud droplets was performed at summit of a mountain (680 m) in Yasaka. To analyze the ambient individual aerosol particles and individual retained particles in cloud droplet, X-ray fluorescence (XRF) analytical method set at SPring-8 on BL39XU was applied. Analytical result enables us not only to compare the characteristics of individual particles scavenged as CCN and those collected at ground-based site, but also to estimate the influence of long-range transport.

Immunochromatographic Strip Assay for Detection of Cronobacter sakazakii in Pure Culture.

PubMed

Song, Xinjie; Shukla, Shruti; Lee, Gibaek; Kim, Myunghee

2016-11-28

Cronobacter sakazakii ( C. sakazakii ) is a foodborne pathogen, posing a high risk of disease to infants and immunocompromised individuals. In order to develop a quick, easy, and sensitive assay for detecting C. sakazakii , a rabbit anti- C. sakazakii immunoglobulin G (IgG) was developed using sonicated cell protein from C. sakazakii . The developed anti- C. sakazakii (IgG) was of good quality and purity, as well as species-specific. The developed rabbit anti- C. sakazakii IgG was attached to the surface of a sulforhodamine B-encapsulated liposome to form an immunoliposome. A test strip was then prepared by coating goat anti-rabbit IgG onto the control line and rabbit anti- C. sakazakii IgG onto the test line, respectively, of a plastic-backed nitrocellulose membrane. A purple color signal both on the test line and the control line indicated the presence of C. sakazakii in the sample, whereas purple color only on the control line indicated the absence of C. sakazakii in the sample. This immunochromatographic strip assay could produce results in 15 min with a limit of detection of 10 7 CFU/ml in C. sakazakii culture. The immunochromatographic strip assay also showed very good specificity without cross-reactivity with other tested Cronobacter species. Based on these results, the developed immunochromatographic strip assay is efficient for the detection of C. sakazakii and has high potential for on-site detection.

Smartphone-based sensing system using ZnO and graphene modified electrodes for VOCs detection.

PubMed

Liu, Lei; Zhang, Diming; Zhang, Qian; Chen, Xing; Xu, Gang; Lu, Yanli; Liu, Qingjun

2017-07-15

Volatile organic compounds (VOCs) detection is in high demand for clinic treatment, environment monitoring, and food quality control. Especially, VOCs from human exhaled breath can serve as significant biomarkers of some diseases, such as lung cancer and diabetes. In this study, a smartphone-based sensing system was developed for real-time VOCs monitoring using alternative current (AC) impedance measurement. The interdigital electrodes modified with zinc oxide (ZnO), graphene, and nitrocellulose were used as sensors to produce impedance responses to VOCs. The responses could be detected by a hand-held device, sent out to a smartphone by Bluetooth, and reported with concentration on an android program of the smartphone. The smartphone-based system was demonstrated to detect acetone at concentrations as low as 1.56ppm, while AC impedance spectroscopy was used to distinguish acetone from other VOCs. Finally, measurements of the exhalations from human being were carried out to obtain the concentration of acetone in exhaled breath before and after exercise. The results proved that the smartphone-based system could be applied on the detection of VOCs in real settings for healthcare diagnosis. Thus, the smartphone-based system for VOCs detection provided a convenient, portable and efficient approach to monitor VOCs in exhaled breath and possibly allowed for early diagnosis of some diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and partial purification of pollen allergens from Artemisia princeps.

PubMed

Park, H S; Hong, C S; Choi, H J; Hahm, K S

1989-12-01

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

PubMed

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

PubMed

Lin, Tong; Shao, Jun-jun; Du, Jun-zheng; Cong, Guo-zheng; Gao, Shan-dian; Chang, Huiyun

2011-09-01

In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 μL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

PubMed Central

Reznick, A Z; Rosenfelder, L; Shpund, S; Gershon, D

1985-01-01

A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins. Images PMID:3898080

Evidence for glucocorticoid receptor binding to a site(s) in a remote region of the 5' flanking sequences of the human proopiomelanocortin gene

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

Tully, D.B.; Hillman, D.; Herbert, E.

1986-05-01

Glucocorticoids negatively regulate expression of the human proopiomelanocortin (POMC) gene. It has been postulated that this effect may be modulated by a direct interaction of the glucocorticoid receptor (GR) with DNA in the vicinity of the POMC promoter. In order to investigate interactions of GR with POMC DNA, DNA-cellulose competitive binding assays have been performed using isolated fragments of cloned POMC DNA to compete with calf thymus DNA-cellulose for binding of triamcinolone acetonide affinity-labelled GR prepared from HeLa S/sub 3/ cells. In these assays, two fragments isolated from the 5' flanking sequences of POMC DNA (Fragment 3,-1765 to -677 andmore » Fragment 4, -676 to +125 with respect to the mRNA cap site) have competed favorably, with Fragment 3 consistently competing more strongly than Fragment 4. Additional studies have been conducted utilizing a newly developed South-western Blot procedure in which specific /sup 32/P-labelled DNA fragments are allowed to bind to dexamethasone mesylate labelled GR immobilized on nitrocellulose filters. Results from these studies have also shown preferential binding by POMC DNA fragments 3 and 4. DNA footprinting and gene transfer experiments are now being conducted to further characterize the nature of GR interaction with POMC DNA.« less

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

PubMed

Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

PubMed

Kang, Keren; Wu, Peidian; Li, Wenmei; Tang, Shixing; Wang, Jihua; Luo, Xiaochun; Xie, Mingquan

2015-01-01

To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

Determination of trace alkaline phosphatase by solid-substrate room-temperature phosphorimetry based on Triticum vulgare lectin labeled with fullerenol.

PubMed

Liu, Jia-Ming; Gao, Fei; Huang, Hong-Hua; Zeng, Li-Qing; Huang, Xiao-Mei; Zhu, Guo-Hui; Li, Zhi-Ming

2008-04-01

Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.

Warning signals for eruptive events in spreading fires

PubMed Central

Fox, Jerome M.; Whitesides, George M.

2015-01-01

Spreading fires are noisy (and potentially chaotic) systems in which transitions in dynamics are notoriously difficult to predict. As flames move through spatially heterogeneous environments, sudden shifts in temperature, wind, or topography can generate combustion instabilities, or trigger self-stabilizing feedback loops, that dramatically amplify the intensities and rates with which fires propagate. Such transitions are rarely captured by predictive models of fire behavior and, thus, complicate efforts in fire suppression. This paper describes a simple, remarkably instructive physical model for examining the eruption of small flames into intense, rapidly moving flames stabilized by feedback between wind and fire (i.e., “wind–fire coupling”—a mechanism of feedback particularly relevant to forest fires), and it presents evidence that characteristic patterns in the dynamics of spreading flames indicate when such transitions are likely to occur. In this model system, flames propagate along strips of nitrocellulose with one of two possible modes of propagation: a slow, structured mode, and a fast, unstructured mode sustained by wind–fire coupling. Experimental examination of patterns in dynamics that emerge near bifurcation points suggests that symptoms of critical slowing down (i.e., the slowed recovery of the system from perturbations as it approaches tipping points) warn of impending transitions to the unstructured mode. Findings suggest that slowing responses of spreading flames to sudden changes in environment (e.g., wind, terrain, temperature) may anticipate the onset of intense, feedback-stabilized modes of propagation (e.g., “blowup fires” in forests). PMID:25675491

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

PubMed Central

Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-01-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

Minimized virus binding for tests of barrier materials.

PubMed Central

Lytle, C D; Routson, L B

1995-01-01

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens. PMID:7574603

Seal and whale meat: two newly recognized food allergies.

PubMed

Moore, Laura M; Rathkopf, Melinda McNeal; Sanner, Carol J; Whisman, Bonnie A; Demain, Jeffrey G

2007-01-01

Alaska's marine mammals compose a large portion of the diet of indigenous coastal Alaskan people. Bowhead whales (Balaena mysticetus) and bearded seals (Erignathus barbatus), inhabitants of the Bering and Beaufort seas along Alaska's western and northern coasts, are 2 of the most important subsistence species, serving as major food sources to the native population. To describe an Inupiaq boy with symptoms consistent with an IgE-mediated food allergy after ingestion of bowhead whale and bearded seal meat. Extracts of cooked bowhead whale and bearded seal were prepared, lyophilized, and evaluated for protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed for each extract, followed by transfer to nitrocellulose and IgE immunoblots. Skin prick testing was conducted using reconstituted extracts of 1:10 wt/vol dilution. Immunoblots revealed serum specific IgE binding with the extracts of bowhead whale and bearded seal meat. Protein bands of approximately 25, 40, 50, and 90 kDa were found in the seal meat. Protein bands of 55 and 90 kDa were found in the whale meat. Skin prick test results were positive to whale and seal extracts with appropriate positive and negative controls. Ten control subjects had negative reactions to both extracts. A patient with moderate anaphylaxis to bowhead whale and bearded seal meat demonstrated serum specific IgE by means of immunoblot and positive skin prick test results. This is the first known reported case of specific IgE to these species.

Visual detection of copper(II) ions in blood samples by controlling the leaching of protein-capped gold nanoparticles.

PubMed

Lee, Yen-Fei; Deng, Ting-Wei; Chiu, Wei-Jane; Wei, Tsao-Yen; Roy, Prathik; Huang, Chih-Ching

2012-04-21

We have developed a simple, low-cost, paper-based probe for the selective colorimetric detection of copper ions (Cu(2+)) in aqueous solutions. The bovine serum albumin (BSA)-modified 13.3-nm Au nanoparticle (BSA-Au NP) probe was designed to detect Cu(2+) ions using lead ions (Pb(2+)) and 2-mercaptoethanol (2-ME) as leaching agents in a glycine-NaOH (pH 12.0) solution. In addition, a nitrocellulose membrane (NCM) was used to trap the BSA-Au NPs, leading to the preparation of a nanocomposite film consisting of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs probe operates on the principle that Cu deposition on the surface of the BSA-Au NPs inhibits their leaching ability, which is accelerated by Pb(2+) ions in the presence of 2-ME. Under optimal solution conditions (5 mM glycine-NaOH (pH 12.0), Pb(2+) (50 μM), and 2-ME (1.0 M)), the Pb(2+)/2-ME-BSA-Au NPs/NCM enabled the detection of Cu(2+) at nanomolar concentrations in aqueous solutions by the naked eye with high selectivity (at least 100-fold over other metal ions). In addition, this cost-effective probe allowed for the rapid and simple determination of Cu(2+) ions in not only natural water samples but also in a complex biological sample (in this case, blood sample).

Facile One-pot Transformation of Iron Oxides from Fe2O3 Nanoparticles to Nanostructured Fe3O4@C Core-Shell Composites via Combustion Waves

PubMed Central

Shin, Jungho; Lee, Kang Yeol; Yeo, Taehan; Choi, Wonjoon

2016-01-01

The development of a low-cost, fast, and large-scale process for the synthesis and manipulation of nanostructured metal oxides is essential for incorporating materials with diverse practical applications. Herein, we present a facile one-pot synthesis method using combustion waves that simultaneously achieves fast reduction and direct formation of carbon coating layers on metal oxide nanostructures. Hybrid composites of Fe2O3 nanoparticles and nitrocellulose on the cm scale were fabricated by a wet impregnation process. We demonstrated that self-propagating combustion waves along interfacial boundaries between the surface of the metal oxide and the chemical fuels enabled the release of oxygen from Fe2O3. This accelerated reaction directly transformed Fe2O3 into Fe3O4 nanostructures. The distinctive color change from reddish-brown Fe2O3 to dark-gray Fe3O4 confirmed the transition of oxidation states and the change in the fundamental properties of the material. Furthermore, it simultaneously formed carbon layers of 5–20 nm thickness coating the surfaces of the resulting Fe3O4 nanoparticles, which may aid in maintaining the nanostructures and improving the conductivity of the composites. This newly developed use of combustion waves in hybridized nanostructures may permit the precise manipulation of the chemical compositions of other metal oxide nanostructures, as well as the formation of organic/inorganic hybrid nanostructures. PMID:26902260

A Sensitive and Rapid Method to Determin the Adhesion Capacity of Probiotics and Pathogenic Microorganisms to Human Gastrointestinal Mucins.

PubMed

Ringot-Destrez, Bélinda; D'Alessandro, Zéa; Lacroix, Jean-Marie; Mercier-Bonin, Muriel; Léonard, Renaud; Robbe-Masselot, Catherine

2018-05-29

Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus to have lasting positive or deleterious effects. The question of which mucin-harboured glycan motifs favour the adhesion of specific microorganisms remains very poorly studied. In the current study, a simple test based on the detection of fluorescent-labeled microorganisms raised against microgram amounts of mucins spotted on nitrocellulose was developed. The adhesion of various probiotic, commensal and pathogenic microorganisms was evaluated on a panel of human purified gastrointestinal mucins and compared with that of commercially available pig gastric mucins (PGM) and of mucins secreted by the colonic cancer cell line HT29-MTX. The latter two proved to be very poor indicators of adhesion capacity on intestinal mucins. Our results show that the nature of the sialylated cores of O -glycans, determined by MALDI MS-MS analysis, potentially enables sialic acid residues to modulate the adhesion of microorganisms either positively or negatively. Other identified factors affecting the adhesion propensity were O -glycan core types and the presence of blood group motifs. This test should help to select probiotics with enhanced adhesion capabilities as well as deciphering the role of specific mucin glycotopes on microbial adhesion.

Assessment of the floral origin of honey by SDS-page immunoblot techniques.

PubMed

Baroni, María V; Chiabrando, Gustavo A; Costa, Cristina; Wunderlin, Daniel A

2002-03-13

We report on the development of a novel alternative method for the assessment of floral origin in honey samples based on the study of honey proteins using immunoblot assays. The main goal of our work was to evaluate the use of honey proteins as chemical markers of the floral origin of honey. Considering that honeybee proteins should be common to all types of honey, we decided to verify the usefulness of pollen proteins as floral origin markers in honey. We used polyclonal anti-pollen antibodies raised in rabbits by repeated immunization of Sunflower (Elianthus annuus) and Eucalyptus (Eucalyptus sp.) pollen extracts. The IgG fraction was purified by immunoaffinity. These antibodies were verified with nitrocellulose blotted pollen and unifloral honey protein extracts. The antibodies anti-Sunflower pollen, bound to the 36 and 33 kDa proteins of Sunflower unifloral honey and to honey containing Sunflower pollen; and the antibodies anti-Eucalyptus sp. pollen bound to the 38 kDa proteins of Eucalyptus sp. unifloral honey in immunoblot assays. Satisfactory results were obtained in differentiating between the types of pollen analyzed and between Sunflower honey and Eucalyptus honey with less cross reactivity with other types of honey from different origin and also with good sensitivity in the detection. This immunoblot method opens an interesting field for the development of new antibodies from different plants, which could serve as an alternative or complementary method to the usual melissopalynological analysis to assess honey floral origin.

Giant Peak Voltage of Thermopower Waves Driven by the Chemical Potential Gradient of Single-Crystalline Bi2 Te3.

PubMed

Singh, Swati; Mun, Hyeona; Lee, Sanghoon; Kim, Sung Wng; Baik, Seunghyun

2017-09-01

The self-propagating exothermic chemical reaction with transient thermovoltage, known as the thermopower wave, has received considerable attention recently. A greater peak voltage and specific power are still demanded, and materials with greater Seebeck coefficients have been previously investigated. However, this study employs an alternative mechanism of transient chemical potential gradient providing an unprecedentedly high peak voltage (maximum: 8 V; average: 2.3 V) and volume-specific power (maximum: 0.11 W mm -3 ; average: 0.04 W mm -3 ) using n-type single-crystalline Bi 2 Te 3 substrates. A mixture of nitrocellulose and sodium azide is used as a fuel, and ultraviolet photoelectron spectroscopy reveals a significant downshift in Fermi energy (≈5.09 eV) of the substrate by p-doping of the fuel. The induced electrical potential by thermopower waves has two distinct sources: the Seebeck effect and the transient chemical potential gradient. Surprisingly, the Seebeck effect contribution is less than 2.5% (≈201 mV) of the maximum peak voltage. The right combination of substrate, fuel doping, and anisotropic substrate geometry results in an order of magnitude greater transient chemical potential gradient (≈5.09 eV) upon rapid removal of fuel by exothermic chemical reaction propagation. The role of fuel doping and chemical potential gradient can be viewed as a key mechanism for enhanced heat to electric conversion performance. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Warning signals for eruptive events in spreading fires.

PubMed

Fox, Jerome M; Whitesides, George M

2015-02-24

Spreading fires are noisy (and potentially chaotic) systems in which transitions in dynamics are notoriously difficult to predict. As flames move through spatially heterogeneous environments, sudden shifts in temperature, wind, or topography can generate combustion instabilities, or trigger self-stabilizing feedback loops, that dramatically amplify the intensities and rates with which fires propagate. Such transitions are rarely captured by predictive models of fire behavior and, thus, complicate efforts in fire suppression. This paper describes a simple, remarkably instructive physical model for examining the eruption of small flames into intense, rapidly moving flames stabilized by feedback between wind and fire (i.e., "wind-fire coupling"-a mechanism of feedback particularly relevant to forest fires), and it presents evidence that characteristic patterns in the dynamics of spreading flames indicate when such transitions are likely to occur. In this model system, flames propagate along strips of nitrocellulose with one of two possible modes of propagation: a slow, structured mode, and a fast, unstructured mode sustained by wind-fire coupling. Experimental examination of patterns in dynamics that emerge near bifurcation points suggests that symptoms of critical slowing down (i.e., the slowed recovery of the system from perturbations as it approaches tipping points) warn of impending transitions to the unstructured mode. Findings suggest that slowing responses of spreading flames to sudden changes in environment (e.g., wind, terrain, temperature) may anticipate the onset of intense, feedback-stabilized modes of propagation (e.g., "blowup fires" in forests).

Rapid Detection of Listeria by Bacteriophage Amplification and SERS-Lateral Flow Immunochromatography

PubMed Central

Stambach, Nicholas R.; Carr, Stephanie A.; Cox, Christopher R.; Voorhees, Kent J.

2015-01-01

A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 106 pfu·mL−1, offering detection below that obtainable by the naked eye (LOD 6 × 107 pfu·mL−1). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 107 colony forming units (cfu)·mL−1, 5 × 106 cfu·mL−1, 5 × 105 cfu·mL−1 and 5 × 104 cfu·mL−1 was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 105 pfu·mL−1) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively. PMID:26694448

Filter Membrane Effects on Water-Extractable Phosphorus Concentrations from Soil.

PubMed

Norby, Jessica; Strawn, Daniel; Brooks, Erin

2018-03-01

To accurately assess P concentrations in soil extracts, standard laboratory practices for monitoring P concentrations are needed. Water-extractable P is a common analytical test to determine P availability for leaching from soils, and it is used to determine best management practices. Most P analytical tests require filtration through a filter membrane with 0.45-μm pore size to distinguish between particulate and dissolved P species. However, filter membrane type is rarely specified in method protocols, and many different types of membranes are available. In this study, three common filter membrane materials (polyether sulfone, nylon, and nitrocellulose), all with 0.45-μm pore sizes, were tested for analytical differences in total P concentrations and dissolved reactive P (DRP) concentrations in water extracts from six soils sampled from two regions. Three of the extracts from the six soil samples had different total P concentrations for all three membrane types. The other three soil extracts had significantly different total P results from at least one filter membrane type. Total P concentration differences were as great as 35%. The DRP concentrations in the extracts were dependent on filter type in five of the six soil types. Results from this research show that filter membrane type is an important parameter that affects concentrations of total P and DRP from soil extracts. Thus, membrane type should be specified in soil extraction protocols. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

nES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins - Separation, Detection, and Sampling of Noncovalent Biospecific Complexes

NASA Astrophysics Data System (ADS)

Engel, Nicole Y.; Weiss, Victor U.; Marchetti-Deschmann, Martina; Allmaier, Günter

2017-01-01

In order to better understand biological events, lectin-glycoprotein interactions are of interest. The possibility to gather more information than the mere positive or negative response for interactions brought mass spectrometry into the center of many research fields. The presented work shows the potential of a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) to detect weak, noncovalent, biospecific interactions besides still unbound glycoproteins and unreacted lectins without prior liquid phase separation. First results for Sambucus nigra agglutinin, concanavalin A, and wheat germ agglutinin and their retained noncovalent interactions with glycoproteins in the gas phase are presented. Electrophoretic mobility diameters (EMDs) were obtained by nES GEMMA for all interaction partners correlating very well with molecular masses determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the individual molecules. Moreover, EMDs measured for the lectin-glycoprotein complexes were in good accordance with theoretically calculated mass values. Special focus was laid on complex formation for different lectin concentrations and binding specificities to evaluate the method with respect to results obtained in the liquid phase. The latter was addressed by capillary electrophoresis on-a-chip (CE-on-a-chip). Of exceptional interest was the fact that the formed complexes could be sampled according to their size onto nitrocellulose membranes after gas-phase separation. Subsequent immunological investigation further proved that the collected complex actually retained its native structure throughout nES GEMMA analysis and sampling.

Skin blotting: a noninvasive technique for evaluating physiological skin status.

PubMed

Minematsu, Takeo; Horii, Motoko; Oe, Makoto; Sugama, Junko; Mugita, Yuko; Huang, Lijuan; Nakagami, Gojiro; Sanada, Hiromi

2014-06-01

The skin performs important structural and physiological functions, and skin assessment represents an important step in identifying skin problems. Although noninvasive techniques for assessing skin status exist, no such techniques for monitoring its physiological status are available. This study aimed to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted proteins from inside the skin following overhydration in mice. The applicability of this technique was further investigated in a clinical setting. Skin blotting involves 2 steps: collecting proteins by attaching a damp nitrocellulose membrane to the surface of the skin, and immunostaining the collected proteins. The authors implanted fluorescein-conjugated dextran (F-DEX)-containing agarose gels into mice and detected the tissue distribution of F-DEX under different blotting conditions. They also analyzed the correlations between inflammatory cytokine secretion and leakage following ultraviolet irradiation in mice and in relation to body mass index in humans. The F-DEX in mice was distributed in the deeper and shallower layers of skin and leaked through the transfollicular and transepidermal routes, respectively. Ultraviolet irradiation induced tumor necrosis factor secretion in the epidermis in mice, which was detected by skin blotting, whereas follicular tumor necrosis factor was associated with body mass index in obese human subjects. These results support the applicability of skin blotting for skin assessment. Skin blotting represents a noninvasive technique for assessing skin physiology and has potential as a predictive and diagnostic tool for skin disorders.

Warning signals for eruptive events in spreading fires

DOE PAGES

Fox, Jerome M.; Whitesides, George M.

2015-02-09

Spreading fires are noisy (and potentially chaotic) systems in which transitions in dynamics are notoriously difficult to predict. As flames move through spatially heterogeneous environments, sudden shifts in temperature, wind, or topography can generate combustion instabilities, or trigger self-stabilizing feedback loops, that dramatically amplify the intensities and rates with which fires propagate. Such transitions are rarely captured by predictive models of fire behavior and, thus, complicate efforts in fire suppression. This study describes a simple, remarkably instructive physical model for examining the eruption of small flames into intense, rapidly moving flames stabilized by feedback between wind and fire (i.e., “wind–firemore » coupling”—a mechanism of feedback particularly relevant to forest fires), and it presents evidence that characteristic patterns in the dynamics of spreading flames indicate when such transitions are likely to occur. Here, in this model system, flames propagate along strips of nitrocellulose with one of two possible modes of propagation: a slow, structured mode, and a fast, unstructured mode sustained by wind–fire coupling. Experimental examination of patterns in dynamics that emerge near bifurcation points suggests that symptoms of critical slowing down (i.e., the slowed recovery of the system from perturbations as it approaches tipping points) warn of impending transitions to the unstructured mode. Lastly, findings suggest that slowing responses of spreading flames to sudden changes in environment (e.g., wind, terrain, temperature) may anticipate the onset of intense, feedback-stabilized modes of propagation (e.g., “blowup fires” in forests).« less

Warning signals for eruptive events in spreading fires

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

Fox, Jerome M.; Whitesides, George M.

Spreading fires are noisy (and potentially chaotic) systems in which transitions in dynamics are notoriously difficult to predict. As flames move through spatially heterogeneous environments, sudden shifts in temperature, wind, or topography can generate combustion instabilities, or trigger self-stabilizing feedback loops, that dramatically amplify the intensities and rates with which fires propagate. Such transitions are rarely captured by predictive models of fire behavior and, thus, complicate efforts in fire suppression. This study describes a simple, remarkably instructive physical model for examining the eruption of small flames into intense, rapidly moving flames stabilized by feedback between wind and fire (i.e., “wind–firemore » coupling”—a mechanism of feedback particularly relevant to forest fires), and it presents evidence that characteristic patterns in the dynamics of spreading flames indicate when such transitions are likely to occur. Here, in this model system, flames propagate along strips of nitrocellulose with one of two possible modes of propagation: a slow, structured mode, and a fast, unstructured mode sustained by wind–fire coupling. Experimental examination of patterns in dynamics that emerge near bifurcation points suggests that symptoms of critical slowing down (i.e., the slowed recovery of the system from perturbations as it approaches tipping points) warn of impending transitions to the unstructured mode. Lastly, findings suggest that slowing responses of spreading flames to sudden changes in environment (e.g., wind, terrain, temperature) may anticipate the onset of intense, feedback-stabilized modes of propagation (e.g., “blowup fires” in forests).« less

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system.

PubMed

Hodgson, A J; Penke, B; Erdei, A; Chubb, I W; Somogyi, P

1985-03-01

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

PubMed

Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-11-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients.

Identification of Optimal Epitopes for Plasmodium falciparum Rapid Diagnostic Tests That Target Histidine-Rich Proteins 2 and 3

PubMed Central

Lee, Nelson; Gatton, Michelle L.; Pelecanos, Anita; Bubb, Martin; Gonzalez, Iveth; Bell, David; Cheng, Qin

2012-01-01

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs. PMID:22259210

Cryptococcus neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucleotide-stimulated macrophages in a fashion independent of capsular polysaccharides.

PubMed

Xiao, Gang; Miyazato, Akiko; Inden, Ken; Nakamura, Kiwamu; Shiratori, Kohei; Nakagawa, Kiyotaka; Miyazawa, Teruo; Suzuki, Kazuo; Kaku, Mitsuo; Kawakami, Kazuyoshi

2008-03-01

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG-ODN protect mice from infection with this fungal pathogen by inducing IFN-gamma. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH-S, an alveolar macrophage cell line, was stimulated with CpG-ODN in the presence of IFN-gamma. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH-S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-kappaB in a luciferase reporter assay with HEK293T cells, although the TLR4-mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG-ODN and IFN-gamma-stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.

Development of a fluorescent immnunochromatographic assay for the procalcitonin detection of clinical patients in China.

PubMed

Wang, Haiying; Wang, Hong; Chen, Shaopei; Dzakah, Emmanuel E; Kang, Keren; Wang, Jihua; Wang, Jufang

2015-04-15

Procalcitonin (PCT) has been recognized as a biomarker in severe inflammation, infection and sepsis. PCT detection in serum requires sensitive and specific antibodies. In this study, we generated monoclonal antibodies (mAbs) and developed fluorescent immunochromatographic assay for PCT detection. Human recombinant PCT was used as immunogen. mAbs against PCT were developed and applied to fluorescent immunochromatographic assay for PCT detection in clinical samples. Out of 35 hybridoma cell lines secreting antibodies against the recombinant PCT, five sensitive and specific cell lines were selected and designated as F6, G2, C2, D2 and E5. All these antibodies have no cross reaction with calcitonin or calcitonin gene-related peptides (CGRP). After screening for pairing, mAb F6 was labeled with fluorescent microspheres and C2 was coated on a nitrocellulose membrane for immunochromatographic test. All 35 clinical samples were detected by the mAb F6-C2 test strips and the bioMérieux PCT assay. The test strips showed high specificity and sensitivity for PCT. Good correlation was observed between our immunochromatographic test strips and the bioMérieux PCT assay (R(2):0.986). These newly developed anti-PCT mAbs and fluorescent immunochromatographic assay can serve as important diagnostic tools for a fast, reliable and point-of-care testing for easy determination of PCT in serum and diagnosis of bacterial infection, inflammation or sepsis. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid enumeration of low numbers of moulds in tea based drinks using an automated system.

PubMed

Tanaka, Kouichi; Yamaguchi, Nobuyasu; Baba, Takashi; Amano, Norihide; Nasu, Masao

2011-01-31

Aseptically prepared cold drinks based on tea have become popular worldwide. Contamination of these drinks with harmful microbes is a potential health problem because such drinks are kept free from preservatives to maximize aroma and flavour. Heat-tolerant conidia and ascospores of fungi can survive pasteurization, and need to be detected as quickly as possible. We were able to rapidly and accurately detect low numbers of conidia and ascospores in tea-based drinks using fluorescent staining followed by an automated counting system. Conidia or ascospores were inoculated into green tea and oolong tea, and samples were immediately filtered through nitrocellulose membranes (pore size: 0.8 μm) to concentrate fungal propagules. These were transferred onto potato dextrose agar and incubated for 23 h at 28 °C. Fungi germinating on the membranes were fluorescently stained for 30 min. The stained mycelia were counted selectively within 90s using an automated counting system (MGS-10LD; Chuo Electric Works, Osaka, Japan). Very low numbers (1 CFU/100ml) of conidia or ascospores could be rapidly counted, in contrast to traditional labour intensive techniques. All tested mould strains were detected within 24h while conventional plate counting required 72 h for colony enumeration. Counts of slow-growing fungi (Cladosporium cladosporioides) obtained by automated counting and by conventional plate counting were close (r(2) = 0.986). Our combination of methods enables counting of both fast- and slow-growing fungi, and should be useful for microbiological quality control of tea-based and also other drinks. Copyright © 2011 Elsevier B.V. All rights reserved.

Point-of-care coagulation monitoring: first clinical experience using a paper-based lateral flow diagnostic device.

PubMed

Hegener, Michael A; Li, Hua; Han, Daewoo; Steckl, Andrew J; Pauletti, Giovanni M

2017-09-01

Vitamin K antagonists such as warfarin are the most widely used class of oral anticoagulants. Due to a narrow therapeutic window, patients on warfarin require regular monitoring. Self-testing using point-of-care (POC) diagnostic devices is available, but cost makes this monitoring method beyond reach for many. The main objective of this research was to assess the clinical utility of a low-cost, paper-based lateral flow POC diagnostic device developed for anticoagulation monitoring without the need for a separate electronic reader. Custom-fabricated lateral flow assay (LFA) test strips comprised of a glass fiber sample pad, a nitrocellulose analytical membrane, a cellulose wicking pad, and a plastic backing card were assembled in a plastic cassette. Healthy volunteers and patients on warfarin therapy were recruited for this prospective study. For each participant, a whole blood sample was collected via fingerstick to determine: (1) international normalized ratio (INR) using the CoaguChek® XS coagulometer, (2) hematocrit by centrifugation, and (3) red blood cell (RBC) travel distance on the experimental LFA device after 240 s using digital image analysis. RBC travel distance measured on the LFA device using blood samples obtained from warfarin patients positively correlated with increasing INR value and the LFA device had the capability to statistically distinguish between healthy volunteer INR values and those for patients groups with INR ≥ 2.6. From these data, it is predicted that this low-cost, paper-based LFA device can have clinical utility for identifying anticoagulated patients taking vitamin K antagonists who are outside of the desired therapeutic efficacy window.

Investigation of gunshot residue patterns using milli-XRF-techniques: first experiences in casework

NASA Astrophysics Data System (ADS)

Schumacher, Rüdiger; Barth, Martin; Neimke, Dieter; Niewöhner, Ludwig

2010-06-01

The investigation of gunshot residue (GSR) patterns for shooting range estimation is usually based on visualizing the lead, copper, or nitrocellulose distributions on targets like fabric or adhesive tape by chemographic color tests. The method usually provides good results but has its drawbacks when it comes to the examination of ammunition containing lead-free primers or bloody clothing. A milli-X-ray fluorescence (m-XRF) spectrometer with a large motorized stage can help to circumvent these problems allowing the acquisition of XRF mappings of relatively large areas (up to 20 x 20 cm) in millimeter resolution within reasonable time (2-10 hours) for almost all elements. First experiences in GSR casework at the Forensic Science Institute of the Bundeskriminalamt (BKA) have shown, that m-XRF is a useful supplementation for conventional methods in shooting ranges estimation, which helps if there are problems in transferring a GSR pattern to secondary targets (e.g. bloody or stained garments) or if there is no suitable color test available for the element of interest. The resulting elemental distributions are a good estimate for the shooting range and can be evaluated by calculating radial distributions or integrated count rates of irregular shaped regions like pieces of human skin which are too small to be investigated with a conventional WD-XRF spectrometer. Beside a mapping mode the milli-XRF offers also point and line scan modes which can also be utilized in gunshot crime investigations as a quick survey tool to identify bullet holes based on the elements present in the wipe ring.

Synthesis of a new energetic nitrate ester

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

Chavez, David E

2008-01-01

Nitrate esters have been known as useful energetic materials since the discovery of nitroglycerin by Ascanio Sobrero in 1846. The development of methods to increase the safety and utility of nitroglycerin by Alfred Nobel led to the revolutionary improvement in the utility of nitroglycerin in explosive applications in the form of dynamite. Since then, many nitrate esters have been prepared and incorporated into military applications such as double-based propellants, detonators and as energetic plasticizers. Nitrate esters have also been shown to have vasodilatory effects in humans and thus have been studied and used for treatments of ailments such as angina.more » The mechanism of the biological response towards nitrate esters has been elucidated recently. Interestingly, many of the nitrate esters used for military purposes are liquids (ethylene glycol dinitrate, propylene glycol dinitrate, etc). Pentaerythritol tetranitrate (PETN) is one of the only solid nitrate esters, besides nitrocellulose, that is used in any application. Unfortunately, PETN melting point is above 100 {sup o}C, and thus must be pressed as a solid for detonator applications. A more practical material would be a melt-castable explosive, for potential simplification of manufacturing processes. Herein we describe the synthesis of a new energetic nitrate ester (1) that is a solid at ambient temperatures, has a melting point of 85-86 {sup o}C and has the highest density of any known nitrate ester composed only of carbon, hydrogen, nitrogen and oxygen. We also describe the chemical, thermal and sensitivity properties of 1 as well as some preliminary explosive performance data.« less

Flexibility of myosin attachment to surfaces influences F-actin motion.

PubMed Central

Winkelmann, D A; Bourdieu, L; Ott, A; Kinose, F; Libchaber, A

1995-01-01

We have analyzed the dependence of actin filament sliding movement on the mode of myosin attachment to surfaces. Monoclonal antibodies (mAbs) that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain (LC2) located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. This method of attachment provides a means of controlling the flexibility and density of myosin on the surface. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these mAbs, and the sliding movement of fluorescently labeled actin filaments was analyzed by video microscopy. Each of these antibodies produced stable myosin-coated surfaces that supported uniform motion of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM mAbs yielded significantly higher velocities (10 microns/s at 30 degrees C) than attachment through anti-LC2 (4-5 microns/s at 30 degrees C). For each antibody, we observed a characteristic value of the myosin density for the onset of F-actin motion and a second critical density for velocity saturation. The specific mode of attachment influences the velocity of actin filaments and the characteristic surface density needed to support movement. Images FIGURE 1 FIGURE 4 FIGURE 8 PMID:7544167

Smartphone-based immunosensor for CA125 detection.

PubMed

Hosu, Oana; Ravalli, Andrea; Lo Piccolo, Giuseppe Mattia; Cristea, Cecilia; Sandulescu, Robert; Marrazza, Giovanna

2017-05-01

In this work, we report the design, the development and the characterization of the analytical performances of a colorimetric smartphone-based immunosensor for the detection of cancer antigen 125 (CA125). The immunosensor was based on a sandwich strategy in which the primary antibody was immobilized by spotting onto the 3D nitrocellulose membrane. The immunospots were subsequently incubated with CA125 solutions, followed by the affinity reaction with a secondary antibody labeled with gold nanoparticles (AuNPs). The antibody-AuNPs captured onto immunospots induced the silver deposition from a silver enhancer solution leading to the formation of gold-silver nanoparticles of different grey color spots depending on CA125 concentration. The 8 megapixels smartphone camera was integrated in a home-made dark box and used as transducer of color image acquisition and data handling. The pixel intensity of the captured images was determined by an image processing algorithm. The experimental parameters involved in each step of the immunosensor design were studied and optimized, obtaining a limit of detection of 30U/mL CA125. The selectivity of the immunoassay was proven against different concentration solutions of Vascular Endothelial Growth Factor (VEGF) antigen as an unspecific protein when a blank signal was obtained for all tested solutions. Finally, preliminary experiments in human serum samples spiked with CA125 protein were also performed. Therefore, the proposed system could represent a powerful point-of-care tool for the next generation technology for detecting and monitoring cancer biomarkers at early stages by taking advantage of nowadays gadgets with enhanced features such as smartphones. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of a major polypeptide of the nuclear pore complex

PubMed Central

1982-01-01

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes. PMID:7153248

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum.

PubMed

Barisone, Gustavo A; Albertali, Isabel E; Sánchez, Mercedes; Cabada, Marcelo O

2003-02-11

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE.

The envelopes of amphibian oocytes: physiological modifications in Bufo arenarum

PubMed Central

Barisone, Gustavo A; Albertali, Isabel E; Sánchez, Mercedes; Cabada, Marcelo O

2003-01-01

A characterization of the Amphibian Bufo arenarum oocyte envelope is presented. It was made in different functional conditions of the oocyte: 1) when it has been released into the coelomic cavity during ovulation (surrounded by the coelomic envelope, (CE), 2) after it has passed through the oviduct and is deposed (surrounded by the viteline envelope, (VE), and 3) after oocyte activation (surrounded by the fertilization envelope, (FE). The characterization was made by SDS-PAGE followed by staining for protein and glycoproteins. Labeled lectins were used to identify glycosidic residues both in separated components on nitrocellulose membranes or in intact oocytes and embryos. Proteolytic properties of the content of the cortical granules were also analyzed. After SDS-PAGE of CE and VE, a different protein pattern was observed. This is probably due to the activity of a protease present in the pars recta of the oviduct. Comparison of the SDS-PAGE pattern of VE and FE showed a different mobility for one of the glycoproteins, gp75. VE and FE proved to have different sugar residues in their oligosaccharide chains. Mannose residues are only present in gp120 of the three envelopes. N-acetyl-galactosamine residues are present in all of the components, except for gp69 in the FE. Galactose residues are present mainly in gp120 of FE. Lectin-binding assays indicate the presence of glucosamine, galactose and N-acetyl galactosamine residues and the absence (or non-availability) of N-acetyl-glucosamine or fucose residues on the envelopes surface. The cortical granule product (CGP) shows proteolytic activity on gp75 of the VE. PMID:12694627

Determination of trace glucose and forecast of human diseases by affinity adsorption solid substrate room temperature phosphorimetry based on Triticum valgaris lectin labeled with 4.0-generation dendrimers

NASA Astrophysics Data System (ADS)

Li, Zhiming; Zhu, Guohui; Liu, Jiaming; Lu, Qiaomei; Yang, Minlan; Wu, Hong; Shi, Xiumei; Chen, Xinhua

2007-08-01

A new phosphorescence labeling reagent Triton-100X-4.0G-D (4.0G-D refers to 4.0-generation dendrimers) was found. Quantitative specific affinity adsorption (AA) reaction between Triton-100X-4.0G-D-WGA and glucose (G) was carried out on the surface of nitrocellulose membrane (NCM), and the Δ Ip of the product of AA reaction was linear correlation to the content of G. Based on the facts above, a new method for the determination of trace G was established by WGA labeled with Triton-100X-4.0G-D affinity adsorption solid substrate room temperature phosphorimetry (Triton-100X-4.0G-D-WGA-AA-SS-RTP). This research showed that AA-SS-RTP for either direct method or sandwich method could combine very well the characteristics of both the high sensitivity of SS-RTP and the specificity of the AA reaction. Detection limits (LD) were 0.24 fg spot -1 for direct method and 0.18 fg spot -1 for sandwich method, indicating both of them were of high sensitivity. The method has been applied to the determination of the content of G in human serum, and the results were coincided with those obtained by glucose oxidize enzyme method. It can also be applied to forecast accurately some human diseases, such as primary hepatic carcinoma, cirrhosis, acute and chronic hepatitis, transfer hepatocellular, etc. Meanwhile, the mechanism for the determination of G with AA-SS-RTP was discussed.

Photolysis of RDX and nitroglycerin in the context of military training ranges.

PubMed

Bordeleau, Geneviève; Martel, Richard; Ampleman, Guy; Thiboutot, Sonia

2013-09-01

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and nitroglycerin (NG) are two energetic materials commonly found in the environment on military training ranges. They are deposited on the ground in the form of solid particles, which can then dissolve in infiltration water or in surface water bodies. The objective of this study was to evaluate whether photolysis by sunlight can significantly contribute to the natural attenuation of RDX and NG (as solid particles or dissolved in surface water) at mid-northern latitudes, where training ranges of Canada and many European countries are located. Experiments conducted at 46.9°N show that both compounds are degraded by sunlight when dissolved in water, with half-lives between 1 and 120d, depending on the compound and time of year. Numerical models may be useful in predicting such photolysis rates, but the models should take into account current ozone levels, as older radiation datasets, collected before the ozone depletion observed since the late 1970s, underestimate the RDX/NG photolysis rate. For solid RDX or NG-bearing particles, photolysis is slower (half-lives of 2-4months), but the degradation rate is still rapid enough to make this process significant in a natural attenuation context. However, photolysis of NG embedded within solid propellant particles cannot proceed to completion, due to the stable nitrocellulose matrix of the propellant. Nonetheless, photolysis clearly constitutes an important attenuation mechanism that should be considered in conceptual models and included in numerical modeling efforts. Copyright © 2013 Elsevier Ltd. All rights reserved.

RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

PubMed Central

Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

2008-01-01

Background Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. Results In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. Conclusion The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis. PMID:19102773

Separation of an associated 90K heat shock protein from the glucocorticoid receptor complex

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

Miller-Diener, A.; Kirsch, T.; Grove, B.

1986-05-01

A 90K heat shock protein(HSP), observed to copurify with the glucocorticoid receptor(GR), can be separated from the complex by 2 methods, allowing investigation of the role of HSP on kinase activity that was previously reported to be inherent to purified activated GR. Na/sub 2/MoO/sub 4/ stabilized unactivated rat hepatic GR complexes have been purified to >10,000-fold using a purification scheme that involves batchwise treatment of cytosol with phosphocellulose/DNAcellulose, elution from an affinity resin, gel filtration and ion exchange chromatography. Samples were subjected to 10-20% gradient SDS-PAGE. Proteins were transferred to nitrocellulose and blotted against monoclonal antibodies to GR(3A6), HSP ormore » nonspecific IgM/G. Immunoblots indicated that HSP was separated from unactivated GR complexes at the affinity step prior to elution of GR with active steroid. GR eluted from the resin with /sup 3/H Triamcinolone acetonide or /sup 3/H Dexamethasone mesylate had an apparent M/sub r/ = 94-96,000 for the steroid binding subunit and is recognized by 3A6. Purification of GR minus the affinity step resulted in copurification of HSP throughout the procedure. However, after Sephadex G75 filtration and subsequent incubation at 25/sup 0/C, 30 min., HSP was separated from activated (DNA binding) GR on DEAE cellulose-52. HSP did not enhance or inhibit /sup 32/P incorporation of the 94K steroid binding subunit nor did it affect phosphorylation of histones by GR.« less

A novel dynamic flow immunochromatographic test (DFICT) using gold nanoparticles for the serological detection of Toxoplasma gondii infection in dogs and cats.

PubMed

Jiang, Wei; Liu, Yingchun; Chen, Yongjun; Yang, Qiufeng; Chun, Peter; Yao, Kailing; Han, Xiangan; Wang, Shaohui; Yu, Shengqing; Liu, Yongjie; Wang, Quan

2015-10-15

A novel dynamic flow immunochromatographic test (DFICT) is proposed for rapid assay utilizing Toxoplasma gondii as a model. The test is based on a proprietary technology that combines the principles of immunochromatography and fluid dynamics. Gold nanoparticles conjugated to staphylococcal protein A (SPA) were prepared in liquid form and used as signal vehicles. T. gondii-specific recombinant antigens and SPA were sprayed onto a nitrocellulose membrane in strips at positions designated as T and C, respectively. The DFICT is performed by applying a 100 µL aliquot of liquid gold-SPA conjugate to the reagent hole and a 5 μL aliquot of serum sample to the sample hole. The results were observable within 5 min by the naked eye. The lowest detectable limit of the assay was determined as the highest dilution (1:320) of positive serum. No cross-reaction of the antibodies with other related canine or feline pathogens was observed. The DFICT can be stored for 12 months at 4°C or 6 months with no loss of sensitivity or specificity. A high degree of consistency was observed between the DFICT and the standard ELISA kit, supporting the reliability of the novel test strip. The introduction of a liquid gold nanoparticle conjugate reagent provides this method with several attractive characteristics, such as ease of manufacture, low sample volume requirements, high selectivity and high efficiency. This method opens a novel pathway for rapid diagnostic screening and field analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Design and development of a field applicable gold nanosensor for the detection of luteinizing hormone.

PubMed

Zambre, Ajit; Chanda, Nripen; Prayaga, Sudhirdas; Almudhafar, Rosana; Afrasiabi, Zahra; Upendran, Anandhi; Kannan, Raghuraman

2012-11-06

In this paper, we describe a novel strategy for the fabrication of a nanosensor for detecting luteinizing hormone (LH) of sheep using a gold nanoparticle-peptide conjugate. A new peptide sequence "CDHPPLPDILFL" (leutinizing hormone peptide, LHP) has been identified, using BLAST and Clustal W analysis, to detect antibody of LH (sheep). LHP has been synthesized and characterized, and their affinity toward anti-LH was established using enzyme linked immunosorbant assay (ELISA) technique. The thiol group in LHP directly binds with gold nanoparticles (AuNPs) to yield AuNP-LHP construct. Detailed physicochemical analysis of AuNP-LHP construct was determined using various analytical techniques. Nanosensor using gold nanoparticle peptide conjugate was developed on the basis of competitive binding of AuNP-LHP and LH toward anti-LH. Nitrocellulose membrane, precoated with anti-LH, was soaked in the mixture of AuNP-LHP and sample of analysis (LH). In the absence of LH (sheep), anti-LH coated on the membrane binds with AuNP-LHP, leading to a distinctive red color, while in the presence of LH, no color appeared in the membrane due to the interaction of anti-LH with LH thereby preventing the binding of AuNP-LHP with membrane bound anti-LH. The sensor assay developed in this study can detect LH (sheep) up to a minimal concentration of ∼50 ppm with a high degree of reproducibility and selectivity. The gold-nanoparticle-peptide based nanosensor would be a simple, portable, effective, and low cost technique for infield applications.

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

PubMed Central

Fariña Sarasqueta, Arantza; Moerland, Elna; de Bruyne, Hanneke; de Graaf, Henk; Vrancken, Tamara; van Lijnschoten, Gesina; van den Brule, Adriaan J.C.

2011-01-01

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2–specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives. PMID:21354055

Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

PubMed

Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

2008-06-22

Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats.

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.

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility

NASA Astrophysics Data System (ADS)

Phillips, Nancy J.; John, Constance M.; Jarvis, Gary A.

2016-07-01

Lipooligosaccharides (LOS) are major microbial virulence factors displayed on the outer membrane of rough-type Gram-negative bacteria. These amphipathic glycolipids are comprised of two domains, a core oligosaccharide linked to a lipid A moiety. Isolated LOS samples are generally heterogeneous mixtures of glycoforms, with structural variability in both domains. Traditionally, the oligosaccharide and lipid A components of LOS have been analyzed separately following mild acid hydrolysis, although important acid-labile moieties can be cleaved. Recently, an improved method was introduced for analysis of intact LOS by MALDI-TOF MS using a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose. In addition to molecular ions, the spectra show in-source "prompt" fragments arising from regiospecific cleavage between the lipid A and oligosaccharide domains. Here, we demonstrate the use of traveling wave ion mobility spectrometry (TWIMS) for IMS-MS and IMS-MS/MS analyses of intact LOS from Neisseria spp. ionized by MALDI. Using IMS, the singly charged prompt fragments for the oligosaccharide and lipid A domains of LOS were readily separated into resolved ion plumes, permitting the extraction of specific subspectra, which led to increased confidence in assigning compositions and improved detection of less abundant ions. Moreover, IMS separation of precursor ions prior to collision-induced dissociation (CID) generated time-aligned, clean MS/MS spectra devoid of fragments from interfering species. Incorporating IMS into the profiling of intact LOS by MALDI-TOF MS exploits the unique domain structure of the molecule and offers a new means of extracting more detailed information from the analysis.

Near-infrared fluorescence-based multiplex lateral flow immunoassay for the simultaneous detection of four antibiotic residue families in milk.

PubMed

Chen, Yiqiang; Chen, Qian; Han, Miaomiao; Liu, Jiangyang; Zhao, Peng; He, Lidong; Zhang, Yuan; Niu, Yiming; Yang, Wenjun; Zhang, Liying

2016-05-15

In this study, we developed a novel near-infrared fluorescence based multiplex lateral flow immunoassay by conjugating a near-infrared label to broad-specificity monoclonal antibody/receptor as detection complexes. Different antigens were dispensed onto separate test zones of nitrocellulose membrane to serve as capture reagents. This assay format allowed the simultaneous detection of four families of antibiotics (β-lactams, tetracyclines, quinolones and sulfonamides) in milk within 20 min. Qualitative and quantitative analysis of target antibiotics were realized by imaging the fluorescence intensity of the near-infrared label captured on respective test lines. For qualitative analysis, the cut-off values of β-lactams, tetracyclines, quinolones and sulfonamides were determined to be 8 ng/mL, 2 ng/mL, 4 ng/mL and 8 ng/mL respectively, which were much lower than the conventional gold nanoparticle based lateral flow immunoassay. For quantitative analysis, the detection ranges were 0.26-3.56 ng/mL for β-lactams, 0.04-0.98 ng/mL for tetracyclines, 0.08-2.0 ng/mL for quinolones, and 0.1-3.98 ng/mL for sulfonamides, with linear correlation coefficients higher than 0.97. The mean spiked recoveries ranged from 93.7% to 108.2% with coefficient of variations less than 16.3%. These results demonstrated that this novel immunoassay is a promising approach for rapidly screening the four families of antibiotic residues in milk. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of a human antigen specific helper factor

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

Richardson, B.

1986-03-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with /sup 35/S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEMmore » line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants.« less

Binding of phosphoinositide-specific phospholipase C-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructured cluster of basic residues.

PubMed

Nomikos, Michail; Mulgrew-Nesbitt, Anna; Pallavi, Payal; Mihalyne, Gyongyi; Zaitseva, Irina; Swann, Karl; Lai, F Anthony; Murray, Diana; McLaughlin, Stuart

2007-06-01

Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.

Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis.

PubMed

Marangoni, Antonella; Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

2016-05-01

Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Antigenic cross-reactivity between Schistosoma mansoni and peanut: a role for cross-reactive carbohydrate determinants (CCDs) and implications for the hygiene hypothesis.

PubMed

Igetei, Joseph E; El-Faham, Marwa; Liddell, Susan; Doenhoff, Michael J

2017-04-01

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti-schistosome sera. A pair of cross-reactive peanut molecules at ~30 000-33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti-S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α-1 and κ-5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α-1 or κ-5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross-reactivities. The results are consistent with the antigenic cross-reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross-reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on 'blocking antibodies' could provide an insight for the inverse relationship observed between schistosome infection and allergies. © 2017 John Wiley & Sons Ltd.

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Patterns of Viral DNA Integration in Cells Transformed by Wild Type or DNA-Binding Protein Mutants of Adenovirus Type 5 and Effect of Chemical Carcinogens on Integration

PubMed Central

Dorsch-Häsler, Karoline; Fisher, Paul B.; Weinstein, I. Bernard; Ginsberg, Harold S.

1980-01-01

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5ts125 and H5ts107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C0t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5ts125 or H5ts107 at either the semipermissive (36°C) or nonpermissive (39.5°C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases XbaI, EcoRI, or HindIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol. 98: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration. Images PMID:6246266

Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.

PubMed Central

Winkelmann, D. A.; Bourdieu, L.; Kinose, F.; Libchaber, A.

1995-01-01

We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement. PMID:7787107

Thermopower Wave-Driven Hybrid Supercapacitor Charging System.

PubMed

Shin, Dongjoon; Hwang, Hayoung; Yeo, Taehan; Seo, Byungseok; Choi, Wonjoon

2016-11-16

The development of new energy sources and harvesting methods has increased with the rapid development of multiscale wireless and portable systems. A thermopower wave (TW) is a potential portable energy source that exhibits a high power density. TWs generate electrical energy via the transport of charges inside micro- or nanostructured materials. This transport is induced by self-propagating combustion. Despite the high specific power of TWs, the generation of energy by TWs is transient, making a TW device a one-time use source, which is a critical limitation on the further advancement of this technology. Herein, we first report the development of a hybrid supercapacitor charging system driven by consecutive TWs to accumulate multiple amounts of energy generated by the repetitive combustion of the chemical fuel. In this study, hybrid layers composed of a supercapacitor (poly(vinyl alcohol)/MnO 2 /nickel) and solid fuel layer (nitrocellulose film) were fabricated as one integrated platform. Combustion was initiated by the ignition of the fuel layer, resulting in the production of electrical energy, attributed to the potential difference between two electrodes, and the transport of charges inside one of the electrodes. Electrical energy could simultaneously and directly charge the supercapacitor, and the discharged voltage could be significantly increased in comparison with the voltage level before the application of a TW. Furthermore, the application of multiple TWs in succession in the hybrid supercapacitor charging system successfully allowed for stack voltage amplification, which was synchronized to each TW. The results of this study could be used to understand the underlying phenomena for charging supercapacitors with the variation of thermal energy and to advance the application of TWs as more efficient, practical energy sources.

G-quadruplex induced stabilization by 2′-deoxy-2′-fluoro-d-arabinonucleic acids (2′F-ANA)

PubMed Central

Peng, Chang Geng; Damha, Masad J.

2007-01-01

The impact of 2′-deoxy-2′-fluoroarabinonucleotide residues (2′F-araN) on different G-quadruplexes derived from a thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), an anti-HIV phosphorothioate aptamer PS-d(T2G4T2) and a DNA telomeric sequence d(G4T4G4) via UV thermal melting (Tm) and circular dichroism (CD) experiments has been investigated. Generally, replacement of deoxyguanosines that adopt the anti conformation (anti-guanines) with 2′F-araG can stabilize G-quartets and maintain the quadruplex conformation, while replacement of syn-guanines with 2′F-araG is not favored and results in a dramatic switch to an alternative quadruplex conformation. It was found that incorporation of 2′F-araG or T residues into a thrombin-binding DNA G-quadruplex stabilizes the complex (ΔTm up to ∼+3°C/2′F-araN modification); 2′F-araN units also increased the half-life in 10% fetal bovine serum (FBS) up to 48-fold. Two modified thrombin-binding aptamers (PG13 and PG14) show an approximately 4-fold increase in binding affinity to thrombin, as assessed via a nitrocellulose filter binding assay, both with increased thermal stability (∼1°C/2′F-ANA modification increase in Tm) and nuclease resistance (4–7-fold) as well. Therefore, the 2′-deoxy-2′-fluoro-d-arabinonucleic acid (2′F-ANA) modification is well suited to tune (and improve) the physicochemical and biological properties of naturally occurring DNA G-quartets. PMID:17636049

Physical and Chemical Compatibility of Injectable Acetaminophen During Simulated Y-Site Administration

PubMed Central

Anderson, Collin; Boehme, Sabrina; Ouellette, Jacquelyn; Stidham, Chanelle; MacKay, Mark

2014-01-01

Purpose: The physical and chemical compatibility of intravenous acetaminophen with commonly administered injectable medications was evaluated. Methods: Simulated Y-site evaluation was accomplished by mixing 2 mL of acetaminophen (10 mg/mL) with 2 mL of an alternative intravenous medication and subsequently storing the mixture in a polypropylene syringe for 4 hours. The aliquot solutions were visually inspected and evaluated for crystal content at 4 hours by infusing 4 mL of the medication mixture through a 0.45-μm nitrocellulose filter disc. Medication mixtures that were selected for chemical stability testing were analyzed by high-performance liquid chromatography at 0, 1, and 4 hours using a Zorbax Eclipse Plus C18, 4.6 x 100 mm, 3.5-μm column for separation of analytes with subsequent diode-array detection. Medications were considered chemically compatible if the concentrations of all components were >90% of the original concentrations during the 4 hour simulated Y-site compatibility test. Results: U.S. Pharmacopeial Convention (USP) standards for physical particle counts were met for acetaminophen injection (10 mg/mL) when combined with cefoxitin, ceftriaxone, clindamycin, dexamethasone, diphenhydramine, dolasetron, fentanyl, granisetron, hydrocortisone, hydromorphone, ketorolac, meperidine, methylprednisolone, midazolam, morphine, nalbuphine, ondansetron, piperacillin/tazobactam, ranitidine, and vancomycin. Injectable acetaminophen is incompatible with acyclovir and diazepam and therefore should not be administered concomitantly with either of these products. Further testing confirmed the chemical compatibility of acetaminophen with ceftriaxone, diphenhydramine, granisetron, ketorolac, nalbuphine, ondansetron, piperacillin/tazobactam, and vancomycin. Conclusion: All medications tested with acetaminophen were physically compatible except for acyclovir and diazepam. All 8 medications tested for chemical compatibility with acetaminophen were stable over the 4 hour simulated Y-site administration study. PMID:24421562

The adhesion of Pseudomonas aeruginosa to high molecular weight human tear film species corresponds to glycoproteins reactive with Sambucus nigra lectin.

PubMed

Aristoteli, Lina Panayiota; Willcox, Mark D P

2006-11-01

Pseudomonas aeruginosa is a pathogen gaining prevalence in contact lens-related corneal ulcers. Tear outflow protects the ocular surface, where high molecular weight tear glycoproteins bind bacteria for removal from the eye. The purpose of the present study was to identify glycoproteins in human tears involved in the adhesion of ocular P. aeruginosa isolates. Basal human tears were applied to a bacterial adhesion assay involving electrophoretic separation of tear components, transfer to nitrocellulose and incubation with biotin-labelled bacteria. Glycoproteins were further characterised using lectin profiling. The results showed large-dimension agarose gels were imperative for the detection of at least four glycoproteins with a migration >200 kDa, including species not previously identified. P. aeruginosa 6294 preferentially bound to a well-defined glycoprotein near the origin of the gel that, unlike other glycoproteins >200 kDa, reacted with Sambucus nigra lectin (sialic acid alpha2-6) but not WGA lectin (N-acetylglucosamine, sialic acid alpha2-3). Adhesion did not involve free biotin label or hydrophobic interactions. Also, the pre-incubation of separated tear glycoproteins with S. nigra lectin increased subsequent adhesion of 6294 to this tear glycoprotein. The less virulent Paer1 strain showed diffuse adhesion in the S. nigra-reactive region at the gel origin. In conclusion, an overlay adhesion assay was developed that identified slow-migrating sialylated glycoprotein species in human tears preferentially bound by P. aeruginosa ocular strains, and S. nigra lectin seemed to enhance the interaction. The study provides a basis for direct investigation of bacterial adhesion to glycoproteins with an apparent migration >200 kDa in tear fluid.

Transcription boundaries of U1 small nuclear RNA.

PubMed Central

Kunkel, G R; Pederson, T

1985-01-01

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly. Images PMID:2942763

Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat.

PubMed

Urayama, Satoshi; Harada, Yoshito; Nakagawa, Yoko; Ban, Susumu; Akasaka, Mari; Kawasaki, Nana; Sawada, Hitoshi

2008-08-01

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

Measurement of anti-Ascaris IgE antibody levels in tropical allergic patients, using modified ELISA.

PubMed

Lynch, N R; Pérez, M; López, R I; Turner, K J

1987-01-01

The two most common situations in which the determination of serum immunoglobulin E (IgE) levels is of interest are allergic disease and helminthic infection. This is of particular importance in the tropical environment, as helminthiasis possibly influences the expression of allergic reactivity. Because of the low absolute serum levels of IgE, solid-phase radioimmunoassay (RIA) is conventionally used for its measurement. The radioactive and toxic volatile reagents required restricted application of such assays in the tropical situation. We evaluated a nitrocellulose-based, avidin biotin-amplified enzyme-linked immunosorbent assay (ELISA) for IgE, in which monoclonal anti-IgE antibodies were employed. Excellent correlations were obtained between ELISA and RIA for both total and allergen-specific IgE measurement. The ELISA was then applied to determine the levels of anti-Ascaris antibodies in selected allergic patients, in whom no cutaneous immediate hypersensitivity reactions were demonstrated against common environmental allergens such as house dust, but who had positive skin reactions to Ascaris extract. When compared with non-allergic subjects who had equivalent cutaneous reactivity, no significant differences were found in total IgE levels, house-dust specific IgE levels or non-reaginic anti-Ascaris antibody levels. However, higher levels of IgE antibody against the parasite were detected in the allergic subjects. This observation raises the question of the possible role of Ascaris infection in the stimulation of allergic reactions in such patients. We describe an immunoenzymatic assay for total and specific IgE antibody that is better adapted to the tropical situation than the commonly used radioimmunoassays.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct measurement for organic solvents diffusion using ultra-sensitive optical resonator

NASA Astrophysics Data System (ADS)

Ali, Amir R.; Elias, Catherine M.

2017-06-01

In this paper, novel techniques using ultra-sensitive chemical optical sensor based on whispering gallery modes (WGM) are proposed through two different configurations. The first one will use a composite micro-sphere, when the solvent interacts with the polymeric optical sensors through diffusion the sphere start to swallow that solvent. In turn, that leads to change the morphology and mechanical properties of the polymeric spheres. Also, these changes could be measured by tracking the WGM shifts. Several experiments were carried out to study the solvent induced WGM shift using microsphere immersed in a solvent atmosphere. It can be potentially used for sensing the trace organic solvents like ethanol and methanol. The second configuration will use a composite beam nitrocellulose composite (NC) structure that acts as a sensing element. In this configuration, a beam is anchored to a substrate in one end, and the other end is compressing the polymeric sphere causing a shift in its WGM. When a chemical molecule is attached to the beam, the resonant frequency of the cantilever will be changed for a certain amount. By sensing this certain resonant frequency change, the existence of a single chemical molecule can be detected. A preliminary experimental model is developed to describe the vibration of the beam structure. The resonant frequency change of the cantilever due to attached mass is examined imperially using acetone as an example. Breath diagnosis can use this configuration in diabetic's diagnosis. Since, solvent like acetone concentration in human breath leads to a quick, convenient, accurate and painless breath diagnosis of diabetics. These micro-optical sensors have been examined using preliminary experiments to fully investigate its response. The proposed chemical sensor can achieve extremely high sensitivity in molecular level.

Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. pruni in symptomatic field samples

PubMed Central

López-Soriano, Pablo; Noguera, Patricia; Gorris, María Teresa; Puchades, Rosa; Maquieira, Ángel; Marco-Noales, Ester; López, María M.

2017-01-01

Xanthomonas arboricola pv. pruni is a quarantine pathogen and the causal agent of the bacterial spot disease of stone fruits and almond, a major threat to Prunus species. Rapid and specific detection methods are essential to improve disease management, and therefore a prototype of a lateral flow immunoassay (LFIA) was designed for the detection of X. arboricola pv. pruni in symptomatic field samples. It was developed by producing polyclonal antibodies which were then combined with carbon nanoparticles and assembled on nitrocellulose strips. The specificity of the LFIA was tested against 87 X. arboricola pv. pruni strains from different countries worldwide, 47 strains of other Xanthomonas species and 14 strains representing other bacterial genera. All X. arboricola pv. pruni strains were detected and cross-reactions were observed only with four strains of X. arboricola pv. corylina, a hazelnut pathogen that does not share habitat with X. arboricola pv. pruni. The sensitivity of the LFIA was assessed with suspensions from pure cultures of three X. arboricola pv. pruni strains and with spiked leaf extracts prepared from four hosts inoculated with this pathogen (almond, apricot, Japanese plum and peach). The limit of detection observed with both pure cultures and spiked samples was 104 CFU ml-1. To demonstrate the accuracy of the test, 205 samples naturally infected with X. arboricola pv. pruni and 113 samples collected from healthy plants of several different Prunus species were analyzed with the LFIA. Results were compared with those obtained by plate isolation and real time PCR and a high correlation was found among techniques. Therefore, we propose this LFIA as a screening tool that allows a rapid and reliable diagnosis of X. arboricola pv. pruni in symptomatic plants. PMID:28448536

[Western Blot diagnostic yield for simultaneous antibody-detection in patients with human cysticercosis, hydatidosis, and human fascioliasis].

PubMed

Davelois, Kelly; Escalante, Hermes; Jara, César

2016-01-01

. To determine the diagnostic yield using western blotting to simultaneously detect antibodies in patients with human cysticercosis, hydatidosis, and human fascioliasis. Materials and methods . Cross-sectional study of diagnostic yield assessment. Excretory/secretory antigens were obtained from Taenia solium larvae, Echinococcus granulosus cysts, and the adult flukes of Fasciola hepática, which were then separated using the polyacrylamide gel electrophoresis technique, transferred, and attached to a nitrocellulose membrane to be probed with sera from the patient infected with the three parasites. The sensitivity of the technique was assessed using 300 individual serum samples, 60 pools of two parasites, and 20 pools of three parasites with 75 sera from patients with other parasites, 10 from patients with other diseases, and 15 from patients without parasites. Results . The technique revealed 13 glycoproteins (GP): GP 35, 31, 24, 23, 18, 17, 14, and 13 kDa for cysticercosis; GP 8, 16, and 21 kDa for hydatidosis; and GP 17 and 23 kDa for fascioliasis. The test detected the presence of antibodies with a sensitivity of 96% (95% confidence interval [CI] = 94.62-98.54%) in the detection of one or the thirteen bands, a specificity of 100% (95% CI = 99.50-100.00%); individually, there was a sensitivity for cysticercosis of 97% (95% CI = 93.16-100.00%), for hydatidosis of 94% (95% CI = 88.85-99.15%) and for fascioliasis of 96% (95% CI = 91.66-100.00%). Conclusions . Western blotting is effective in the simultaneous detection of antibodies in patients with human cysticercosis, hydatidosis, and fascioliasis, and it can be used as a diagnostic test to either rule out or confirm the presence of antibodies in endemic areas.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein.

PubMed

Chao, Kinlin L; Kulakova, Liudmila; Herzberg, Osnat

2017-02-14

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn's disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88 DNVD 91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

PubMed Central

Chao, Kinlin L.; Kulakova, Liudmila; Herzberg, Osnat

2017-01-01

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn’s disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88DNVD91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport. PMID:28154144

Annexin II is associated with mRNAs which may constitute a distinct subpopulation.

PubMed Central

Vedeler, A; Hollås, H

2000-01-01

Protein-mRNA interactions affect mRNA transport, anchorage, stability and translatability in the cytoplasm. During the purification of three subpopulations of polysomes, it was observed that a 36-kDa protein, identified as annexin II, is associated with only one specific population of polysomes, namely cytoskeleton-associated polysomes. This association appears to be calcium-dependent since it was sensitive to EGTA and could be reconstituted in vitro. UV irradiation resulted in partial, EGTA-resistant cross-linking of annexin II to the polysomes. Binding of (32)P-labelled total RNA to proteins isolated from the cytoskeleton-bound polysomes on a NorthWestern blot resulted in a radioactive band having the same mobility as annexin II and, most importantly, purified native annexin II immobilized on nitrocellulose specifically binds mRNA. The mRNA population isolated from cytoskeleton-bound polysomes binds to annexin II with the highest affinity as compared with those isolated from free or membrane-bound polysomes. Interestingly, the annexin II complex, isolated from porcine small intestinal microvilli was a far better substrate for mRNA binding than the complex derived from transformed Krebs II ascites cells. When cytoskeleton-associated polysomes were split into 60 S and 40 S ribosomal subunits, and a peak containing mRNA complexes, annexin II fractionated with the mRNAs. Finally, using affinity purification of mRNA on poly(A)(+)-coupled magnetic beads, annexin II was only detected in association with messenger ribonucleoproteins (mRNPs) present in the cytoskeletal fraction (non-polysomal mRNPs). These results, derived from both in vitro experiments and cell fractionation, suggest that annexin II binds directly to the RNA moiety of mRNP complexes containing a specific population of mRNAs. PMID:10839987

Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples.

PubMed

Pengsuk, Chalinan; Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

2013-04-15

A strip test for the detection of Vibrio cholerae O139 was developed using two monoclonal antibodies (MAbs), namely VC-273 and VC-812, which specifically bind to the lipopolysaccharide and capsular polysaccharide of V. cholerae O139. The MAb VC-273 gold nanoparticle conjugate was sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. MAb VC-812 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated as T and C, respectively. The test strips were assessed for their ability to directly detect V. cholerae O139 using samples dispersed in application buffer, and a 100 μL aliquot of sample was applied to the sample chamber. The results were observable within 20 min after application of the sample. In samples containing V. cholerae O139, the antigen was bound to the colloidal gold-conjugated MAb to form an antibody-antigen complex. This complex was captured by the MAbs at the T test line, resulting in the appearance of a reddish-purple band at the T position. The sensitivity of the test was determined to be 10⁴ cfu mL⁻¹. Direct detection of V. cholerae O139 in various fresh seafood samples could be accomplished with similar sensitivities. The detection limit was substantially improved to 1 cfu mL⁻¹ of the original bacterial content after pre-incubation of the sample in alkaline peptone water for 12 h. The V. cholerae strip test provides several advantages over other methods, including the speed and simplicity of use because there is no requirement for sophisticated equipment. Copyright © 2012 Elsevier B.V. All rights reserved.

Facile one-pot transformation using structure-guided combustion waves of micro-nanostructured β-Bi2O3 to α-Bi2O3@C and analysis of electrochemical capacitance

NASA Astrophysics Data System (ADS)

Hwang, Hayoung; Shin, Jung-ho; Lee, Kang Yeol; Choi, Wonjoon

2018-01-01

Precise phase-transformation can facilitate control of the properties of various materials, while an organic coating surrounding inorganic materials can yield useful characteristics. Herein, we demonstrate facile, selective manipulation of micro-nanostructured bismuth oxide (Bi2O3) for phase transformation from microflower-like β-Bi2O3 to micropill-like α-Bi2O3, with carbon-coating layer deposition, using structure-guided combustion waves (SGCWs). Microflower-like β-Bi2O3 are synthesized as core materials and nitrocellulose is coated on their surfaces for the formation of core-shell hybrid structures of Bi2O3 and chemical fuel. The SGCWs, which propagate along the core-material and fuel interfaces, apply high thermal energy (550-600 °C) and deposit incompletely combusted carbonaceous fuel on the microflower-like β-Bi2O3 to enable transformation to α-phase and carbon-coating-layer synthesis. SGCW-induced improvements to the electrochemical characteristics of the developed micropill-like α-Bi2O3@C, compared with the microflower-like β-Bi2O3, are investigated. The enhanced stability from the α-phase Bi2O3 and micropill-like structures during charge-discharge cycling improves the specific capacitance, while the carbon-coating layers facilitate increased electrical conductivity. SGCW-based methods exhibit high potential for selective phase manipulation and synthesis of carbon coatings surrounding micro-nanomaterials. They constitute a low-cost, fast, large-scale process for metal oxides, ceramics, and hybrid materials, implemented through control of the processing parameters by tuning the temperature, chemical fuel, and ambient conditions.

A novel line immunoassay based on recombinant virulence factors enables highly specific and sensitive serologic diagnosis of Helicobacter pylori infection.

PubMed

Formichella, Luca; Romberg, Laura; Bolz, Christian; Vieth, Michael; Geppert, Michael; Göttner, Gereon; Nölting, Christina; Walter, Dirk; Schepp, Wolfgang; Schneider, Arne; Ulm, Kurt; Wolf, Petra; Busch, Dirk H; Soutschek, Erwin; Gerhard, Markus

2013-11-01

Helicobacter pylori colonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome. H. pylori virulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to important H. pylori virulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed in Escherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) were H. pylori negative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), the recomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, the recomLine assay provides a valuable tool for the diagnosis of H. pylori infection.

IgG subclass reactivity to human cardiac myosin in cardiomyopathy patients is indicative of a Th1-like autoimmune disease

PubMed Central

Skyllouriotis, P; Skyllouriotis-Lazarou, M; Natter, S; Steiner, R; Spitzauer, S; Kapiotis, S; Valent, P; Hirschl, A M; Guber, S E; Laufer, G; Wollenek, G; Wolner, E; Wimmer, M; Valenta, R

1999-01-01

Studies performed in mice together with the demonstration of increased levels of heart-specific autoantibodies, cytokines and cytokine receptors in sera from cardiomyopathy (CMP) patients argued for a pathogenic role of autoimmune mechanisms in CMP. This study was designed to analyse the presence of IgG anti-heart antibodies in sera from patients suffering from hypertrophic and dilatative forms of CMP as well as from patients with ischaemic heart disease and healthy individuals. Patients' sera were analysed for IgG reactivity to Western-blotted extracts prepared from human epithelial and endothelial cells, heart and skeletal muscle specimens as well as from Streptococcus pyogenes. The IgG subclass (IgG1–4) reactivity to purified human cardiac myosin was analysed by ELISA. While sera from CMP patients and healthy individuals displayed comparable IgG reactivity to a variety of human proteins, cardiac myosin represented the prominent antigen detected strongly and preferentially by sera from CMP patients. Pronounced IgG anti-cardiac myosin reactivity was frequently found in sera from patients with dilatative CMP and reduced ventricular function. ELISA analyses revealed a prominent IgG2/IgG3 anti-cardiac myosin reactivity in CMP sera, indicating a preferential Th1-like immune response. Elevated anti-cytomegalovirus, anti-enterovirus IgG titres as well as IgG reactivity to nitrocellulose-blotted S. pyogenes proteins were also frequently observed in the group of CMP patients. If further work can support the hypothesis that autoreactivity to cardiac myosin represents a pathogenic factor in CMP, specific immunomodulation of this Th1- towards a Th2-like immune response may represent a promising therapeutic strategy for CMP. PMID:9933448

Phosphine-free synthesis of high-quality reverse type-I ZnSe/CdSe core with CdS/CdxZn1 - xS/ZnS multishell nanocrystals and their application for detection of human hepatitis B surface antigen

NASA Astrophysics Data System (ADS)

Shen, Huaibin; Yuan, Hang; Niu, Jin Zhong; Xu, Shasha; Zhou, Changhua; Ma, Lan; Li, Lin Song

2011-09-01

Highly photoluminescent (PL) reverse type-I ZnSe/CdSe nanocrystals (NCs) and ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS core/multishell NCs were successfully synthesized by a phosphine-free method. By this low-cost, 'green' synthesis route, more than 10 g of high-quality ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS NCs were synthesized in a large scale synthesis. After the overgrowth of a CdS/CdxZn1 - xS/ZnS multishell on ZnSe/CdSe cores, the PL quantum yields (QYs) increased from 28% to 75% along with the stability improvement. An amphiphilic oligomer was used as a surface coating agent to conduct a phase transfer experiment, core/multishell NCs were dissolved in water by such surface modification and the QYs were still kept above 70%. The as-prepared water dispersible ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS core/multishell NCs not only have high fluorescence QYs but also are extremely stable in various physiological conditions. Furthermore, a biosensor system (lateral flow immunoassay system, LFIA) for the detection of human hepatitis B surface antigen (HBsAg) was developed by using this water-soluble core/multishell NCs as a fluorescent label and a nitrocellulose filter membrane for lateral flow. The result showed that such ZnSe/CdSe/CdS/CdxZn1 - xS/ZnS core/multishell NCs were excellent fluorescent labels to detect HBsAg. The sensitivity of HBsAg detection could reach as high as 0.05 ng ml - 1.

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

PubMed

Smeringaiova, Ingrida; Trosan, Peter; Mrstinova, Miluse Berka; Matecha, Jan; Burkert, Jan; Bednar, Jan; Jirsova, Katerina

2017-09-01

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.

Diversity of citrus tristeza virus isolates indicated by dsRNA analysis.

PubMed

Dodds, J A; Jordan, R L; Roistacher, C N; Jarupat, T

1987-01-01

One major dsRNA of molecular weight (MW) 13.3 X 10(6) and two others (MW 1.9 X 10(6) and 0.8 X 10(6] were routinely detected by polyacrylamide gel electrophoresis in extracts from sweet orange (Citrus sinensis) or citron (Citrus medica) infected with each of 66 isolates of citrus tristeza virus (CTV). Several additional dsRNA were also commonly detected, usually as weakly stained bands in reproducible positions in gels, but some were very prominent, e.g., a dsRNA of MW 1.7 X 10(6) associated with a seedling yellows isolate (sy-1). No dsRNA was detected in equivalent extracts from noninoculated sweet orange and citron. End-labeled [32P] probes were made from purified full-length viral RNA or polyacrylamide gel-purified full-length dsRNA of a nonseedling yellows (nsy-1) and a seedling yellows (sy-1) isolate of CTV. Each of the four probes was able to hybridize to all major and most minor dsRNAs of both isolates in composite polyacrylamide/agrarose gels, including the 1.7 X 10(6) dsRNA specific to the seedling yellows isolate, and could readily detect CTV nucleic acid sequences in extracts from bark of infected sweet orange plants spotted onto nitrocellulose membranes. One dsRNA (MW 0.5 X 10(6] was very prominent in some isolates and much less so, or undetectable, in other isolates and 66 isolates have been screened for the presence of this dsRNA. There was a strong correlation between inability to detect the 0.5 X 10(6) dsRNA and the designation of an isolate as neither a seedling yellows type nor a stem pitting isolate of grapefruit; these properties were typical for isolates of CTV from southern California.

Seroprevalence of Fasciola gigantica infection in bovines using cysteine proteinase dot enzyme-linked immunosorbent assay

PubMed Central

Kumar, Niranjan; Varghese, Anju; Solanki, J. B.

2017-01-01

Aim: The objective of the present study was to know the seroprevalence status of Fasciola gigantica infection in cattle and buffaloes using cysteine proteinase (CP) antigen in dot enzyme-linked immunosorbent assay (ELISA) format under field conditions. Materials and Methods: As per the standard protocol, the sera were collected from the blood of 112 cattle and 38 buffaloes of coastal areas of Navsari district, South Gujarat, India. The indirect ELISA was performed on the strip of nitrocellulose paper blotted with 1 µl of CP antigen, to detect F. gigantica seropositive animals. Results: The native CP of F. gigantica revealed a single visible band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no any noted cross-reaction between the selected antigen and sera of Gastrothylax crumenifer-infected animals in ELISA. Out of 150 screened bovines, the sera of 47 (31.33%) were found to be reactive in dot-ELISA, with a prevalence rate of 31.25% and 31.58% in cattle and buffaloes, respectively. The seropositive bovines with heavy, moderate, and light level of infection were 44.68%, 34.04%, and 21.28%, respectively (p<0.05 between heavy and light; p>0.05 between moderate and heavy or light). The share of F. gigantica seropositive and negative animals was 31% and 69%, respectively. The optical density at 450 nm of pooled sera of seropositive bovines with heavy, moderate, and light reactivity in plate-ELISA was significantly higher with field or reference ­negative sera. Conclusion: The CP-based dot-ELISA can be useful for field veterinarians for quick and timely isolation of the animals requiring urgent flukicide therapy. PMID:29184364

Visualisation and quantification of intracellular interactions of Neisseria meningitidis and human α-actinin by confocal imaging.

PubMed

Murillo, Isabel; Virji, Mumtaz

2010-10-24

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors(1). This process leads to bacterial invasion of endothelial cells(2). More recently, we observed an interaction of Opc with a 100 kDa protein found in whole cell lysates of human cells(3). We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as α-actinin. As no surface expressed α-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and α-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with α-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Encapsulation of ionic electroactive polymers: reducing the interaction with environment

NASA Astrophysics Data System (ADS)

Jaakson, P.; Aabloo, A.; Tamm, T.

2016-04-01

Ionic electro-active polymer (iEAP) actuators are composite materials that change their mechanical properties in response to external electrical stimulus. The interest in these devices is mainly driven by their capability to generate biomimetic movements, and their potential use in soft robotics. The driving voltage of an iEAP-actuator (0.5… 3 V) is at least an order of magnitude lower than that needed for other types of electroactive polymers. To apply iEAP-actuators in potential real-world applications, the capability of operating in different environments (open air, different solvents) must be available. In their natural form, the iEAP-actuators are capable of interacting with the surrounding environment (evaporation of solvent from the electrolyte solution, ion or solvent exchange, humidity effects), therefore, for prevention of unpredictable behavior of the actuator and the contamination of the environment, encapsulation of the actuator is needed. The environmental contamination aspect of the encapsulation material is substantial when selecting an applicable encapsulant. The suitable encapsulant should form thin films, be light in weight, elastic, fit tightly, low cost, and easily reproducible. The main goal of the present study is to identify and evaluate the best potential encapsulation techniques for iEAPactuators. Various techniques like thin film on liquid coating, dip coating, hot pressing, hot rolling; and several materials like polydimethylsiloxane, polyurethane, nitrocellulose, paraffin-composite-films were investigated. The advantages and disadvantages of the combinations of the above mentioned techniques and materials are discussed. Successfully encapsulated iEAP-actuators gained durability and were stably operable for long periods of time under ambient conditions. The encapsulation process also increased the stability of the iEAP-actuator by minimizing the environment effects. This makes controlling iEAP-actuators more straight-forward and reliable since there is no need to take the environmental factors like relative humidity and/or gas circulation into account.

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

PubMed

Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

2007-09-01

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides

PubMed Central

Fujiwara, Yuichiro; Kubo, Yoshihiro

2006-01-01

Phosphoinositides (PIPns) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X2 channels also are regulated by PIPns, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X2 channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-d-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X2 cytoplasmic domain to anionic lipids using PIPns-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP2s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X2 with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIPns play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X2 cytoplasmic C-terminal domain. PMID:16857707

A New Look on Protein-Polyphenol Complexation during Honey Storage: Is This a Random or Organized Event with the Help of Dirigent-Like Proteins?

PubMed Central

Brudzynski, Katrina; Sjaarda, Calvin; Maldonado-Alvarez, Liset

2013-01-01

Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002) with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS –PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT) on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a) high molecular weight complexes (230–180 kDa) enriched in proteins but possessing a limited reducing activity toward the NBT and (b) lower molecular size complexes (110–85 kDa) enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, “protein-type” complexes were formed by protein cross-linking, while in the smaller, “polyphenol-type” complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS) analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the reaction of quinones with proteins and polyphenols could possibly be under assumed guidance of dirigent proteins. PMID:24023654

A MATHEMATICAL ANALYSIS OF SELEX

PubMed Central

Levine, Howard A.; Nilsen-Hamilton, Marit

2007-01-01

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a procedure by which a mixture of nucleic acids can be separated into pure components with the goal of isolating those with specific biochemical activities. The basic idea is to combine the mixture with a specific target molecule and then separate the target-NA complex from the resulting reaction. The target-NA complex is then separated by mechanical means (for example by nitrocellulose filtration), the NA is then eluted from the complex, amplified by PCR (polymerase chain reaction) and the process repeated. After several rounds, one should be left with a pool of [NA]that consists mostly of the species in the original pool that best binds to the target. In Irvine et al. (1991) a mathematical analysis of this process was given. In this paper we revisit Irvine et al. (1991). By rewriting the equations for the SELEX process, we considerably reduce the labor of computing the round to round distribution of nucleic acid fractions. We also establish necessary and sufficient conditions for the SELEX process to converge to a pool consisting solely of the best binding nucleic acid to a fixed target in a manner that maximizes the percentage of bound target. The assumption is that there is a single nucleic acid binding site on the target that permits occupation by no more than one nucleic acid. We analyze the case for which there is no background loss, (no support losses and no free [NA] left on the support.) We then examine the case in which such there are such losses. The significance of the analysis is that it suggests an experimental approach for the SELEX process as defined in Irvine et al. (1991) to converge to a pool consisting of a single best binding nucleic acid without recourse to any a-priori information about the nature of the binding constants or the distribution of the individual nucleic acid fragments. PMID:17218151

Surface-Controlled Properties of Myosin Studied by Electric Field Modulation.

PubMed

van Zalinge, Harm; Ramsey, Laurence C; Aveyard, Jenny; Persson, Malin; Mansson, Alf; Nicolau, Dan V

2015-08-04

The efficiency of dynamic nanodevices using surface-immobilized protein molecular motors, which have been proposed for diagnostics, drug discovery, and biocomputation, critically depends on the ability to precisely control the motion of motor-propelled, individual cytoskeletal filaments transporting cargo to designated locations. The efficiency of these devices also critically depends on the proper function of the propelling motors, which is controlled by their interaction with the surfaces they are immobilized on. Here we use a microfluidic device to study how the motion of the motile elements, i.e., actin filaments propelled by heavy mero-myosin (HMM) motor fragments immobilized on various surfaces, is altered by the application of electrical loads generated by an external electric field with strengths ranging from 0 to 8 kVm(-1). Because the motility is intimately linked to the function of surface-immobilized motors, the study also showed how the adsorption properties of HMM on various surfaces, such as nitrocellulose (NC), trimethylclorosilane (TMCS), poly(methyl methacrylate) (PMMA), poly(tert-butyl methacrylate) (PtBMA), and poly(butyl methacrylate) (PBMA), can be characterized using an external field. It was found that at an electric field of 5 kVm(-1) the force exerted on the filaments is sufficient to overcome the frictionlike resistive force of the inactive motors. It was also found that the effect of assisting electric fields on the relative increase in the sliding velocity was markedly higher for the TMCS-derivatized surface than for all other polymer-based surfaces. An explanation of this behavior, based on the molecular rigidity of the TMCS-on-glass surfaces as opposed to the flexibility of the polymer-based ones, is considered. To this end, the proposed microfluidic device could be used to select appropriate surfaces for future lab-on-a-chip applications as illustrated here for the almost ideal TMCS surface. Furthermore, the proposed methodology can be used to gain fundamental insights into the functioning of protein molecular motors, such as the force exerted by the motors under different operational conditions.

A new look on protein-polyphenol complexation during honey storage: is this a random or organized event with the help of dirigent-like proteins?

PubMed

Brudzynski, Katrina; Sjaarda, Calvin; Maldonado-Alvarez, Liset

2013-01-01

Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002) with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS -PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT) on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a) high molecular weight complexes (230-180 kDa) enriched in proteins but possessing a limited reducing activity toward the NBT and (b) lower molecular size complexes (110-85 kDa) enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, "protein-type" complexes were formed by protein cross-linking, while in the smaller, "polyphenol-type" complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS) analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the reaction of quinones with proteins and polyphenols could possibly be under assumed guidance of dirigent proteins.

A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper.

PubMed

Costa, M N; Veigas, B; Jacob, J M; Santos, D S; Gomes, J; Baptista, P V; Martins, R; Inácio, J; Fortunato, E

2014-03-07

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.

Sensitivity of a rapid point of care assay for early HIV antibody detection is enhanced by its ability to detect HIV gp41 IgM antibodies.

PubMed

Moshgabadi, Noushin; Galli, Rick A; Daly, Amelia C; Ko, Sze Mun Shirley; Westgard, Tayla E; Bulpitt, Ashley F; Shackleton, Christopher R

2015-10-01

Anti-HIV-1 IgM antibody is an important immunoassay target for early HIV antibody detection. The objective of this study is to determine if the early HIV antibody sensitivity of the 60s INSTI test is due to detection of anti-HIV-1 IgM in addition to IgG. To demonstrate HIV gp41 IgM antibody capture by the INSTI HIV-1 gp41 recombinant antigen, an HIV-IgM ELISA was conducted with commercial HIV-1 seroconversion samples. To demonstrate that the INSTI dye-labelled Protein A-based colour developer (CD) has affinity to human IgM, commercial preparations of purified human immunoglobulins (IgM, IgD, IgA, IgE, and IgG) were blotted onto nitrocellulose (NC) and probed with the CD to observe spot development. To determine that INSTI is able to detect anti-HIV-1 IgM antibody, early seroconversion samples, were tested for reduced INSTI test spot intensity following IgM removal. The gp41-based HIV-IgM ELISA results for 6 early seroconversion samples that were INSTI positive determined that the assay signal was due to anti-HIV-1 IgM antibody capture by the immobilised gp41 antigen. The dye-labelled Protein-A used in the INSTI CD produced distinct spots for purified IgM, IgA, and IgG blotted on the NC membrane. Following IgM removal from 21HIV-1 positive seroconversion samples with known or undetermined anti-HIV-1 IgM levels that were western blot negative or indeterminate, all samples had significantly reduced INSTI test spot intensity. The INSTI HIV-1/HIV-2 Antibody Test is shown to detect anti-HIV-1 IgM antibodies in early HIV infection which enhances its utility in early HIV diagnosis. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

A multiplex immunochromatographic test using gold nanoparticles for the rapid and simultaneous detection of four nitrofuran metabolites in fish samples.

PubMed

Wang, Quan; Liu, Yingchun; Wang, Mingyan; Chen, Yongjun; Jiang, Wei

2018-01-01

There is an urgent need for the rapid and simultaneous detection of multiple analytes present in a sample matrix. Here, a multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites, i.e., 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), in fish samples. Four different antigens were separately immobilized in four test lines on a nitrocellulose membrane. Goat anti-mouse immunoglobulin (IgG) was used as a control. Sensitive and specific monoclonal antibodies (mAbs) that recognize the corresponding antigens were selected for the assay, and no cross-reactivity between the antibodies in the detection assay was observed. The free analytes in samples or standards were pre-incubated with freeze-dried mAb-gold conjugates to improve the sensitivity of the detection assay. The multi-ICT detection was accomplished in less than 15 min by the naked eye. The cutoff values for the strip test were 0.5 ng/mL for AOZ and 0.75 ng/mL for AHD, SEM, and AMOZ, which were all below the maximum residue levels set by the European Union and China. A high degree of consistency was observed between the multi-ICT method and commercially available enzyme-linked immunosorbent assay (ELISA) kits using spiked, incurred, and "blind" fish samples, indicating the accuracy, reproducibility, and reliability of the novel test strip. This newly developed multi-ICT strip assay is suitable for the rapid and high-throughput screening of four nitrofuran metabolites in fish samples on-site, with no treatment or devices required. Graphical abstract A multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites (AOZ, SEM, AMOZ, and AHD) in fish samples.

Identification of structural variations in the carboxyl terminus of Alzheimer's disease-associated βA4[1–42] amyloid using a monoclonal antibody

PubMed Central

Jayasena, U L H R; Gribble, S K; Mckenzie, A; Beyreuther, K; Masters, C L; Underwood, J R

2001-01-01

The accumulation of amyloid plaques and amyloid congophilic angiopathy (ACA) in the brains of affected individuals is one of the main pathological features of Alzheimer's disease. Within these deposits, the βA4 (Aß) polypeptide represents a major component with the C-terminal 39–43 amino acid variants being most abundant. Using a mouse IgG1 MoAb produced by hybridoma βA4[35–43]-95.2 3B9, which reacts with the epitope is defined by the amino acid residues βA438[GVV]40, this study has identified a unique conformation within the carboxyl terminus of human βA4[1–42]. Although the βA438[GVV]40 sequence is present within the C-termini of human βA4[1–40] and βA4[1–43] and the βA4-containing region of human APP, the βA4[35–43]-95.2 3B9 MoAb (designated MoAb 3B9) does not bind these polypeptides, demonstrating a high degree of specificity for the βA438[GVV]40 epitope as presented within the βA4[1–42] sequence. The βA4[1–42] epitope bound by MoAb 3B9 is sensitive to heating (100°C for 5 min) and is denatured by SDS but not by oxidative radio-iodination of βA4 or by adsorption to plastic surfaces or nitrocellulose. The recognition of βA4 plaque deposits and ACA by MoAb 3B9 within formalin-fixed sections of human AD brain demonstrates the potential of these antibodies for investigating the role of the unique βA4[1–42] conformation in the development of Alzheimer's disease. PMID:11422208

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 New Method for Blood NT-proBNP Determination Based on a Near-infrared Point of Care Testing Device with High Sensitivity and Wide Scope.

PubMed

Zhang, Xiao Guang; Shu, Yao Gen; Gao, Ju; Wang, Xuan; Liu, Li Peng; Wang, Meng; Cao, Yu Xi; Zeng, Yi

2017-06-01

To develop a rapid, highly sensitive, and quantitative method for the detection of NT-proBNP levels based on a near-infrared point-of-care diagnostic (POCT) device with wide scope. The lateral flow assay (LFA) strip of NT-proBNP was first prepared to achieve rapid detection. Then, the antibody pairs for NT-proBNP were screened and labeled with the near-infrared fluorescent dye Dylight-800. The capture antibody was fixed on a nitrocellulose membrane by a scribing device. Serial dilutions of serum samples were prepared using NT-proBNP-free serum series. The prepared test strips, combined with a near-infrared POCT device, were validated by known concentrations of clinical samples. The POCT device gave the output of the ratio of the intensity of the fluorescence signal of the detection line to that of the quality control line. The relationship between the ratio value and the concentration of the specimen was plotted as a work curve. The results of 62 clinical specimens obtained from our method were compared in parallel with those obtained from the Roche E411 kit. Based on the log-log plot, the new method demonstrated that there was a good linear relationship between the ratio value and NT-proBNP concentrations ranging from 20 pg/mL to 10 ng/mL. The results of the 62 clinical specimens measured by our method showed a good linear correlation with those measured by the Roche E411 kit. The new LFA detection method of NT-proBNP levels based on the near-infrared POCT device was rapid and highly sensitive with wide scope and was thus suitable for rapid and early clinical diagnosis of cardiac impairment. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Development of a quantitative rapid diagnostic test for multibacillary leprosy using smart phone technology.

PubMed

Paula Vaz Cardoso, Ludimila; Dias, Ronaldo Ferreira; Freitas, Aline Araújo; Hungria, Emerith Mayra; Oliveira, Regiane Morillas; Collovati, Marco; Reed, Steven G; Duthie, Malcolm S; Martins Araújo Stefani, Mariane

2013-10-23

Despite efforts to eliminate leprosy as public health problem, delayed diagnosis and disabilities still occur in many countries. Leprosy diagnosis remains based on clinical manifestations and the number of clinicians with expertise in leprosy diagnosis is in decline. We have developed a new immunochromatographic test with the goal of producing a simple and rapid system that can be used, with a minimal amount of training, to provide an objective and consistent diagnosis of multibacillary leprosy. The test immobilizes two antigens that have been recognized as excellent candidates for serologic diagnosis (the PGL-I mimetic, ND-O, and LID-1), on a nitrocellulose membrane. This allows the detection of specific IgM and IgG antibodies within 20 minutes of the addition of patient sera. Furthermore, we coupled the NDO-LID® rapid tests with a new cell phone-based test reader platform (Smart Reader®) to provide objective interpretation that was both quantifiable and consistent. Direct comparison of serologic responses indicated that the rapid test detected a greater proportion of leprosy patients than a lab-based PGL-I ELISA. While positive responses were detected by PGL-I ELISA in 83.3% of multibacillary patients and 15.4% of paucibacillary patients, these numbers were increased to 87% and 21.2%, respectively, when a combination of the NDO-LID® test and Smart Reader® was used. Among multibacillary leprosy the sensitivity of NDO-LID® test assessed by Smart Reader® was 87% (95% CI, 79.2-92.7%) and the specificity was 96.1% (95% CI, 91.7- 98.6%). The positive predictive value and the negative predictive value of NDO-LID® tests were 94% (95% CI, 87.4-97.8%) and 91.4% (95% CI, 85.9-95.2%), respectively. The widespread provision of rapid diagnostic tests to facilitate the diagnosis or prognosis of multibacillary leprosy could impact on leprosy control programs by aiding early detection, directing appropriate treatment and potentially interrupting Mycobacterium leprae transmission.

A multidisciplinary approach to the study of cultural heritage environments: Experience at the Palatina Library in Parma.

PubMed

Pasquarella, C; Balocco, C; Pasquariello, G; Petrone, G; Saccani, E; Manotti, P; Ugolotti, M; Palla, F; Maggi, O; Albertini, R

2015-12-01

The aim of this paper is to describe a multidisciplinary approach including biological and particle monitoring, and microclimate analysis associated with the application of the Computational Fluid Dynamic (CFD). This approach was applied at the Palatina historical library in Parma. Monitoring was performed both in July and in December, in the absence of visitors and operators. Air microbial monitoring was performed with active and passive methods. Airborne particles with a diameter of ≥0.3, ≥0.5, ≥1 and ≥5 μm/m3, were counted by a laser particle counter. The surface contamination of shelves and manuscripts was assessed with nitrocellulose membranes. A spore trap sampler was used to identify both viable and non-viable fungal spores by optical microscope. Microbiological contaminants were analyzed through cultural and molecular biology techniques. Microclimatic parameters were also recorded. An infrared thermal camera provided information on the surface temperature of the different building materials, objects and components. Transient simulation models, for coupled heat and mass-moisture transfer, taking into account archivist and general public movements, combined with the related sensible and latent heat released into the environment, were carried out applying the CFD-FE (Finite Elements) method. Simulations of particle tracing were carried out. A wide variability in environmental microbial contamination, both for air and surfaces, was observed. Cladosporium spp., Alternaria spp., Aspergillus spp., and Penicillium spp. were the most frequently found microfungi. Bacteria such as Streptomyces spp., Bacillus spp., Sphingomonas spp., and Pseudoclavibacter as well as unculturable colonies were characterized by molecular investigation. CFD simulation results obtained were consistent with the experimental data on microclimatic conditions. The tracing and distribution of particles showed the different slice planes of diffusion mostly influenced by the convective airflow. This interdisciplinary research represents a contribution towards the definition of standardized methods for assessing the biological and microclimatic quality of indoor cultural heritage environments. Copyright © 2015 Elsevier B.V. All rights reserved.

Advanced thermopower wave in novel ZnO nanostructures/fuel composite.

PubMed

Lee, Kang Yeol; Hwang, Hayoung; Choi, Wonjoon

2014-09-10

Thermopower wave is a new concept of energy conversion from chemical to thermal to electrical energy, produced from the chemical reaction in well-designed hybrid structures between nanomaterials and combustible fuels. The enhancement and optimization of energy generation is essential to make it useful for future applications. In this study, we demonstrate that simple solution-based synthesized zinc oxide (ZnO) nanostructures, such as nanorods and nanoparticles are capable of generating high output voltage from thermopower waves. In particular, an astonishing improvement in the output voltage (up to 3 V; average 2.3 V) was achieved in a ZnO nanorods-based composite film with a solid fuel (collodion, 5% nitrocellulose), which generated an exothermic chemical reaction. Detailed analyses of thermopower waves in ZnO nanorods- and cube-like nanoparticles-based hybrid composites have been reported in which nanostructures, output voltage profile, wave propagation velocities, and surface temperature have been characterized. The average combustion velocities for a ZnO nanorods/fuel and a ZnO cube-like nanoparticles/fuel composites were 40.3 and 30.0 mm/s, while the average output voltages for these composites were 2.3 and 1.73 V. The high output voltage was attributed to the amplified temperature in intermixed composite of ZnO nanostructures and fuel due to the confined diffusive heat transfer in nanostructures. Moreover, the extended interfacial areas between ZnO nanorods and fuel induced large amplification in the dynamic change of the chemical potential, and it resulted in the enhanced output voltage. The differences of reaction velocity and the output voltage between ZnO nanorods- and ZnO cube-like nanoparticles-based composites were attributed to variations in electron mobility and grain boundary, as well as thermal conductivities of ZnO nanorods and particles. Understanding this astonishing increase and the variation of the output voltage and reaction velocity, precise ZnO nanostructures, will help in formulating specific strategies for obtaining enhanced energy generation from thermopower waves.

A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper

NASA Astrophysics Data System (ADS)

Costa, M. N.; Veigas, B.; Jacob, J. M.; Santos, D. S.; Gomes, J.; Baptista, P. V.; Martins, R.; Inácio, J.; Fortunato, E.

2014-03-01

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.

Dot immunoassay for the simultaneous determination of postvaccination immunity against pertussis, diphtheria, and tetanus.

PubMed

Khramtsov, Pavel; Bochkova, Maria; Timganova, Valeria; Zamorina, Svetlana; Rayev, Mikhail

2017-06-01

A dot immunoassay for simultaneous semiquantitative detection of IgG against tetanus toxoid (Ttx) and diphtheria toxoid (Dtx) and qualitative detection of anti-Bordetella pertussis IgGs in human blood serum using carbon nanoparticles functionalized with streptococcal protein G was developed. Inactivated B. pertussis cells in suspension form were used as an antigen in the immunoassay. Pertussis, tetanus, and diphtheria antigens were separately spotted onto nitrocellulose strips, and then the immunostrips were successively incubated with blood sera and a suspension of carbon nanoparticles. The immunostrips were then scanned with a flatbed scanner, and the images obtained were processed with ImageJ. One hundred fifty-five venous blood serum samples from children vaccinated with diphtheria, tetanus, and whole-cell pertussis (DTwP) vaccine were tested in comparison with a conventional ELISA and agglutination test. The total time required for analysis of 32 serum samples was less than 3 h. Comparison between the results of the dot immunoassay and the corresponding ELISA/agglutination test revealed a high level of agreement (Cohen's kappa between 0.765 and 0.813). The lower limit of quantification was 0.06 IU/ml for anti-Ttx and anti-Dtx. The intra-assay coefficients of variation were less than 15% for anti-Ttx and anti-Dtx and less than 10% for anti-pertussis. The diagnostic sensitivity of detection of the antibody protection level was 93.5% for anti-Ttx [95% confidence interval (CI) 83.5-97.9%], 92.4% for anti-Dtx (95% CI 80.9297.5%), and 90.2% for anti-pertussis (95% CI 75.9-96.8%). The diagnostic specificity was 90.9% for anti-Ttx (95% CI 57.1-99.5%), 85% for anti-Dtx (95% CI 61.1-96.0%), and 89.3% for anti-pertussis (95%CI 80.8-94.5%). The dot immunoassay developed does not require expensive reading equipment, and allows detection of antibodies against three antigens in a single analysis. The immunostrips can be stored for a long time without changes in the coloration of the spots. Graphical Abstract The assay procedure. BC Bordetella pertussis cell suspension, CNP carbon nanoparticle, Dtx diphtheria toxoid, Ttx tetanus toxoid.

Thoracoscopic modified pleural tent for spontaneous pneumothorax.

PubMed

Kawachi, Riken; Matsuwaki, Rie; Tachibana, Keisei; Karita, Shin; Nakazato, Yoko; Tanaka, Ryota; Nagashima, Yasushi; Takei, Hidefumi; Kondo, Haruhiko

2016-08-01

We developed a modified pleural tent (m-tent) procedure and used it in our hospital in almost 30 consecutive patients with spontaneous pneumothorax. The objective of this study was to clarify the feasibility and effectiveness of a thoracoscopic m-tent for the treatment of spontaneous pneumothorax. From July 2013 to November 2014, 107 patients with spontaneous pneumothorax were treated in our institution. Eighty-nine of these patients were analysed retrospectively. The inclusion criteria for thoracoscopic m-tent for spontaneous pneumothorax were multiple and widespread bullae, postoperative relapse and secondary spontaneous pneumothorax. The surgical procedures were usually performed through three ports. After bullectomy, an m-tent is made to strip the parietal pleura off the chest wall from about the level of the fourth or fifth rib to the apex, and two or three ligations are then applied to fix the pleural tent and lung parenchyma. Patients in whom an m-tent was not indicated underwent bullectomy plus coverage using absorbable materials. Twenty-seven patients underwent bullectomy plus m-tent (m-tent group) and 62 underwent bullectomy plus coverage over a staple line using an absorbable material such as a polyglycolic acid sheet or nitrocellulose sheet (coverage group). No severe postoperative complications were observed in either group. The m-tent and coverage groups showed significant differences in operation time (129 vs 86 min, mean), haemorrhage (12.8 vs 7.2 ml), postoperative hospital stay (3.7 vs 2.9 days) and postoperative painkiller intake (8.6 vs 6.8 days). Recurrence was observed in 1 (3.7%) and 2 patients (3.2%), respectively. The thoracoscopic m-tent procedure requires a longer operation, a longer hospital stay and greater painkiller intake. However, these differences are acceptable, and an m-tent should be considered as an option for pleural reinforcement in spontaneous pneumothorax, especially in patients who are complicated with severe pulmonary emphysema, widespread bullae or recurrent pneumothorax. © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Phenotypic characterization of mononuclear inflammatory cells following equine hydroxyapatite/collagen block grafting in rats.

PubMed

Alsuwaiyan, Asim; Wang, Bing-Yan; Cohen, Robert E

2012-12-01

To measure the inflammatory changes associated with the implantation of an equine hydroxyapatite and collagen-containing block graft (eHAC block) in a rodent model system, an eHAC block graft was implanted subcutaneously in rats. Control groups included saline, turpentine oil, and human mineralized particulate allograft (hMPA). Animals were sacrificed and tissue samples obtained after three days, as well as after 1, 2, 4 and 8 weeks. A panel of immunologic probes was used to identify circulatory monocytic cells (ED1), resident mononuclear phagocytes (ED2), mononuclear phagocytes of lymphoid origin (ED3), expression of Ia antigen (OX6), T-cells (OX19), and B-cells (OX33). Immunocytochemical localization was performed and mononuclear cells localized with each immunologic probe counted. Rat sera obtained after eight weeks were used for nitrocellulose dot-blotting to assess circulating anti-equine immunoglobulins. Statistical analysis was performed using two-way analysis of variance, in conjunction with the Bonferroni correction to account for multiple comparisons. A transient increase in monocytes at 3 days and 1 week was observed in all groups, but was significantly higher in the turpentine control (P < 0.0001). A significant increase in the numbers of mononuclear cells detected with clones ED2 and ED3 was observed in specimens from the turpentine group, in contrast to the other groups in the 3 day to 4 week interval (P < 0.0001), as well as within all time periods (P < 0.0001). A statistically significant difference in numbers of ED3-positive cells was observed in the hMPA group compared to the saline and the eHAC block groups after one week (P < 0.0001). Significantly more OX6-positive cells were observed in the turpentine group, compared to other groups (3 days to 1 week; P < 0.0001). T-lymphocytes were essentially absent except for rats given turpentine (after 1 week). No B-lymphocyte response was found and none of the rats developed systemic anti-equine antibodies. These data indicate that a cellular immune response is not elicited following implantation with the eHAC block graft, which might serve as an alternative material for regenerative therapy.

Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection.

PubMed Central

Rapp, V J; Ross, R F

1986-01-01

Sera from pigs infected with Haemophilus (Actinobacillus) pleuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumoniae which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (greater than or equal to 94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000- and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000- and 95,000-molecular-weight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and greater than or equal to 94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from H. pleuropneumoniae serotypes 1 and 7; however, several OMPs and the lipopolysaccharide or polysaccharide determinants of these serotypes appeared to be type specific. Images PMID:3536748

Nocturnal activities and host preferences of Phlebotomus orientalis in extra-domestic habitats of Kafta-Humera lowlands, Kala-azar endemic, Northwest Ethiopia.

PubMed

Lemma, Wossenseged; Tekie, Habte; Abassi, Ibrahim; Balkew, Meshesha; Gebre-Michael, Teshome; Warburg, Alon; Hailu, Asrat

2014-12-17

Phlebotomus orientalis feeds on a variety of wild and domestic animals and transmits Leishmania donovani from hitherto unknown reservoir hosts to humans in extra-domestic habitats in the Metema-Humera lowlands. The aim of this study was to determine the nocturnal activities of P. orientalis and its preferred blood meal hosts. Collections of Phlebotomus orientalis were made by using CDC light traps to determine the density as P. orientalis/hour CDC trap and preference to rodents by using Turner's traps in agricultural fields, animal shelters and thickets of Acacia seyal in Baeker site-1 and Gelanzeraf site-2. The blood meal sources were detected by Reverse Line Blot (RLB) of cytochrome b polymerase chain reaction (PCR) amplification in August, 2012 from collections of sand flies in thickets of A. seyal (March 2011) and dense mixed forest (July 2011) in Baeker site 1. RLB PCR involved first amplification of animal specific sequences of cytochrome b using PCR techniques. Then the amplified sequence was hybridized with 11 species-specific probes for domestic animals adsorbed on nitrocellulose membrane for calorimetric color detection. A total of 6,083 P. orientalis (2,702 males and 3,381 females) were collected at hourly intervals using 22 CDC traps from January to May 2013. The peak activities of P. orientalis were at 1.00 a.m (134.0 ± 7.21) near animal shelters, 3.00 a.m (66.33 ± 46.40) in agricultural fields and 21:00 pm (40.6 ± 30.06) in thickets of A. seyal. This species was not attracted to the different species of rodents in trials carried out in March and April 2013. RLB PCR identified 7 human (28%), 9 mixed (human and cattle) (36%) and 2 cattle (8%) blood meals while 7 were unknown (28%). Female P. orientalis can bite humans in extra-domestic habitats of Kafta-Humera lowlands at any hour of the night with peak biting after midnight.

Electrical characteristics of mammalian cells on porous supports

NASA Astrophysics Data System (ADS)

Chen, Guo

2003-10-01

The quantification of epithelial barrier functions by measuring the trans-epithelial electrical resistance (TER) and using the Electric Cell-substrate Impedance Sensing (ECIS) has been complicated by the current flowing inside the narrow space underneath cells. This thesis work, by examining the electrical characteristics of epithelial cells on porous supports, is aimed to tackle this problem. A mathematical model has been constructed to quantify the impedance from the various sources within a cell/electrode system. This model presents three cell-related parameters, alpha, Rb and Cm: alpha stands for the impedance contribution from the above-mentioned current underneath cells, Rb is an equivalent representation of epithelial barrier functions and Cm denotes the capacitive impedance of cell membranes. Analysis of the three parameters as well as the electrode impedance (Z e) has revealed two experimental approaches to reduce or eliminate the complication of alpha to the deduction of Rb: lowering alpha down to zero or lowering both Ze and alpha. The experimental realization of the first approach has been studied by examining the electrical characteristics of the African green monkey kidney (BS-C-1) and Madin-Darby canine kidney (MDCK-II) cells on porous filters of mixed esters of cellulose or nitrocellulose. A unique setup featuring a plastic/filter/plastic triple-layer structure was constructed to measure the impedance of cells on filters. With the extremely low alpha, all the electrical characteristics can be explained by using an equivalent circuit and Rb can be directly obtained from the resistance difference in the low frequency range. The second approach has been experimentally investigated by examining the electrical characteristics of BS-C-1 cells on porous/rough electrodes, i.e. the gold ECIS electrodes electrochemically coated with conducting polypyrrole/heparin composites or platinum black. Ze and alpha, especially the former, were found to be significantly lowered, which greatly reduces the effect of alpha and yields many new impedance features. Rb can be also directly obtained in a different way from that for the solely lowered alpha on the non-conducting porous filters.

Rod outer segment-associated N-acetylgalactosaminylphosphotransferase.

PubMed

Sweatt, A J; Balsamo, J; Lilien, J

1995-01-01

To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells. Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized. ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass. The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other proteins, but mostly toward very large molecules that may be IPM proteoglycans. It is not yet known whether the enzyme of the adult retina specifically transfers sugar or sugar-phosphate groups to its acceptors. It is proposed that the ROS GalNAcPTase is involved in the modulation of adhesive phenomena between or within photoreceptors or between photoreceptors and the interphotoreceptor matrix.

Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor.

PubMed

Jakhar, Seema; Pundir, C S

2018-02-15

The nanoparticles (NPs) aggregates of commercial urease from jack beans (Canavalia ensiformis) were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine dihydrochloride. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM), UV and Fourier transform infrared (FTIR) spectroscopy. The TEM images of urease NPs showed their size in the range, 18-100nm with an average of 51.2nm. The ENPs were more active and stable with a longer shelf life than native enzyme molecules. The ENPs were immobilized onto chitosan (CHIT) activated nitrocellulose (NC) membrane via glutaraldehyde coupling with 32.22% retention of initial activity of free ureaseNPs with a conjugation yield of 1.63mg/cm 2 . This NC membrane was mounted at the lower/sensitive end of the ammonium ion selective electrode (AISE) with O-ring and then electrode was connected to a digital pH meter to construct a potentiometric urea biosensor. The biosensor exhibited optimum response within 10s at pH 5.5and 40°C. The biosensor was employed for measurement of potentiometric determination of urea in sera of apparently healthy and persons suffering from kidney disorders. The biosensor displayed a low detection limit of 1µM/L with a wide working range of 2-80µM/L (0.002-0.08mM) and sensitivity of 23mV/decade. The analytical recovery of added urea in serum was 106.33%. The within and between-batch coefficient of variations (CVs) of present biosensor were 0.18% and 0.32% respectively. There was a good correlation (r = 0.99) between sera urea values obtained by reference method (Enzymic colorimetric kit method) and the present biosensor. The biosensor had negligible interference from Na + ,K + ,NH +4 and Ca 2+ but Mg 2+ ,Cu 2+ and ascorbic acid but had slight interference, which was overcome by specific ion selective electrode. The ENPs bound NC membrane was used maximally 8-9 times per day over a period of 180 days, when stored in 0.01M sodium acetate buffer pH 5.5 at 4°C. Copyright © 2017 Elsevier B.V. All rights reserved.

Serum immunoglobulin E and immunoglobulin G reactivity to Agaricus bisporus proteins in mushroom cultivation workers.

PubMed

Khakzad, Z; Hedayati, M T; Mahdian, S; Mayahi, S

2015-06-01

Although molds are regarded as the main fungal allergen sources, evidence indicates that spores of Basidiomycota including Agaricus bisporus ( A. bisporus ) can be also found at high concentrations in the environment and may cause as many respiratory allergies as molds. The aim of the present study was to evaluate specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies against A. bisporus via immunoblotting technique in individuals working at mushroom cultivation centers. In this study, 72 workers involved in the cultivation and harvest of button mushrooms were enrolled. For the analysis of serum IgE and IgG, A. bisporus grown in Sabouraud dextrose broth was harvested and ruptured by liquid nitrogen and glass beads. The obtained sample was centrifuged and the supernatant was collected as "crude extract" (CE). CE was separated via Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The separated proteins were transferred to a nitrocellulose filter and the bands responsive to IgE and IgG were identified by anti-human conjugated antibodies. All participants were screened in terms of total IgE level. Among 72 workers, 18 (25%) had a total IgE level higher than 188 IU/mL. In SDS-PAGE, the CE of A. bisporus showed 23 different protein bands with a molecular weight range of 13-80 kDa. The sera of 23.6% and 55.5% of participants showed positive response, with specific IgE and IgG antibodies against A. bisporus in the blot, respectively. The bands with molecular weights of 62 and 68 kDa were the most reactive protein components of A. bisporus to specific IgE antibodies. Moreover, bands with molecular weights of 57 and 62 kDa showed the highest reactivity to IgG, respectively. Also, 62 and 68 kDa components were the most reactive bands with both specific IgG and IgE antibodies. The obtained findings revealed that A. bisporus has different allergens and antigens, which contribute to its potential as an aeroallergen in hypersensitivity-related reactions of the lungs.

The Unculturables: targeted isolation of bacterial species associated with canine periodontal health or disease from dental plaque.

PubMed

Davis, Ian J; Bull, Christopher; Horsfall, Alexander; Morley, Ian; Harris, Stephen

2014-08-01

The current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. A case in point is periodontal disease, which is the most widespread oral disease in dogs. If untreated the disease results in significant pain, eventual loss of the dentition and potentially an increased risk of systemic diseases. Previous molecular based studies have identified the bacterial species associated with periodontal disease in dogs; however without cultured strains from many of these species it has not been possible to study whether they play a role in the disease process. Using a quantitative polymerase chain reaction (qPCR) directed approach a range of microbiological media were screened and optimized to enrich for previously uncultivated target species. A systematic screening methodology was then employed to isolate the species of interest. In cases where the target species were not cultivable in isolation, helper strains grown underneath a nitrocellulose membrane were used to provide the necessary growth factors. This guided media optimization approach enabled the purification of 14 species, 8 of which we had previously been unable to cultivate in isolation. It is also applicable to the targeted isolation of isolates from species that have previously been cultured (for example to study intra-species variation) as demonstrated by the successful isolation of 6 targeted isolates of already cultured species. To our knowledge this is the first time this combination of qPCR guided media optimization, strategic screening and helper strain support has been used successfully to isolate previously uncultured bacteria. This approach can be applied to any uncultured bacterial species where knowledge of their nutritional requirements or low relative abundance impedes their isolation.

Enzyme-linked immunosorbent assays for Z-DNA.

PubMed

Thomas, M J; Strobl, J S

1988-10-01

Dot blot and transblot enzyme-linked immunosorbent assays (e.l.i.s.a.) are described which provide sensitive non-radioactive methods for screening Z-DNA-specific antisera and for detecting Z-DNA in polydeoxyribonucleotides and supercoiled plasmids. In the alkaline phosphatase dot blot e.l.i.s.a., Z-DNA, Br-poly(dG-dC).poly(dG-dC), or B-DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), Br-poly(dI-dC).poly(dI-dC), or salmon sperm DNA were spotted onto nitrocellulose discs and baked. The e.l.i.s.a. was conducted in 48-well culture dishes at 37 degrees C using a rabbit polyclonal antiserum developed against Br-poly(dG-dC).poly(dG-dC), an alkaline phosphatase-conjugated second antibody, and p-nitrophenol as the substrate. Under conditions where antibody concentrations were not limiting, alkaline phosphatase activity was linear for 2 h. Dot blot e.l.i.s.a. conditions are described which allow quantification of Z-DNA [Br-poly(dG-dC).poly(dG-dC)] within the range 5-250 ng. Dot blot and transblot horseradish peroxidase e.l.i.s.a. are described that detect Z-DNA within supercoiled plasmid DNAs immobilized on diazophenylthioether (DPT) paper. In the transblot e.l.i.s.a., plasmid pUC8 derivatives containing 16, 24, or 32 residues of Z-DNA were electrophoresed in agarose gels and electrophoretically transferred to DPT paper. Z-DNA-antibody complexes were detected by the horseradish peroxidase-catalysed conversion of 4-chloro-1-naphthol to a coloured product that was covalently bound to the DPT paper. Z-DNA antibody reactivity was specific for supercoiled Z-DNA containing plasmids after removal of the antibodies cross-reactive with B-DNA by absorption onto native DNA-cellulose. The transblot e.l.i.s.a. was sensitive enough to detect 16 base pairs of alternating G-C residues in 100 ng of pUC8 DNA.

Synthesis, thermolysis, and sensitivities of HMX/NC energetic nanocomposites.

PubMed

Wang, Yi; Song, Xiaolan; Song, Dan; Liang, Li; An, Chongwei; Wang, Jingyu

2016-07-15

1,3,5,7-Tetranittro-1,3,5,7-tetrazocane/nitrocellulose (HMX/NC) nanocomposites were successfully synthesized by an improved sol-gel-supercritical method. NC nanoparticles with a size of ∼30nm were cross-linked to form a network structure, and HMX nanoparticles were imbedded in the nano-NC matrix. The key factors, i.e., the selection of catalyst and solvent, were probed. No phase transformation of the HMX occurred before or after fabrication, and the molecular structures of the HMX and NC did not change. Thermal analyses were performed, and the kinetic and thermodynamic parameters, such as activation energy (EK), per-exponent factor (lnAK), rate constant (k), activation heat (ΔH(≠)), activation free energy (ΔG(≠)), activation entropy (ΔS(≠)), critical temperature of thermal explosion (Tb), and critical heating rate of thermal explosion (dT/dt)Tb, were calculated. The results indicate that HMX/NC presented a much lower activation energy (165.03kJ/mol) than raw HMX (282.5kJ/mol) or raw NC (175.51kJ/mol). The chemical potential (ΔG(≠)) for the thermal decomposition of HMX/NC has a positive value, which means that the activation of the molecules would not proceed spontaneously. The significantly lower ΔH(≠) value of HMX/NC, which represents the heat needed to be absorbed by an explosive molecule to change it from its initial state to an activated state, implies that the molecules of HMX/NC are much easier to be activated than those of raw HMX. Similarly, the HMX/NC presented a much lower Tb (168.2°C) than raw HMX (283.2°C). From the results of the sensitivity tests, the impact and friction sensitivities of HMX/NC were significantly decreased compared with those of raw HMX, but the thermal sensitivity was distinctly higher. The activation of the particles under external stimulation was simulated, and the mechanism was found to be crucial. Combining the thermodynamic parameters, the mechanism as determined from the results of the sensitivity tests was discussed in detail. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a POC Test for TB Based on Multiple Immunodominant Epitopes of M. tuberculosis Specific Cell-Wall Proteins

PubMed Central

Gonzalez, Jesus M.; Francis, Bryan; Burda, Sherri; Hess, Kaitlyn; Behera, Digamber; Gupta, Dheeraj; Agarwal, Ashutosh Nath; Verma, Indu; Verma, Ajoy; Myneedu, Vithal Prasad; Niedbala, Sam; Laal, Suman

2014-01-01

The need for an accurate, rapid, simple and affordable point-of-care (POC) test for Tuberculosis (TB) that can be implemented in microscopy centers and other peripheral health-care settings in the TB-endemic countries remains unmet. This manuscript describes preliminary results of a new prototype rapid lateral flow TB test based on detection of antibodies to immunodominant epitopes (peptides) derived from carefully selected, highly immunogenic M. tuberculosis cell-wall proteins. Peptide selection was initially based on recognition by antibodies in sera from TB patients but not in PPD-/PPD+/BCG-vaccinated individuals from TB-endemic settings. The peptides were conjugated to BSA; the purified peptide-BSA conjugates striped onto nitrocellulose membrane and adsorbed onto colloidal gold particles to devise the prototype test, and evaluated for reactivity with sera from 3 PPD-, 29 PPD+, 15 PPD-unknown healthy subjects, 10 patients with non-TB lung disease and 124 smear-positive TB patients. The assay parameters were adjusted to determine positive/negative status within 15 minutes via visual or instrumented assessment. There was minimal or no reactivity of sera from non-TB subjects with the striped BSA-peptides demonstrating the lack of anti-peptide antibodies in subjects with latent TB and/or BCG vaccination. Sera from most TB patients demonstrated reactivity with one or more peptides. The sensitivity of antibody detection ranged from 28–85% with the 9 BSA-peptides. Three peptides were further evaluated with sera from 400 subjects, including additional PPD-/PPD+/PPD-unknown healthy contacts, close hospital contacts and household contacts of untreated TB patients, patients with non-TB lung disease, and HIV+TB- patients. Combination of the 3 peptides provided sensitivity and specificity>90%. While the final fully optimized lateral flow POC test for TB is under development, these preliminary results demonstrate that an antibody-detection based rapid POC lateral flow test based on select combinations of immunodominant M. tb-specific epitopes may potentially replace microscopy for TB diagnosis in TB-endemic settings. PMID:25247820

The Orientation of Gastric Biopsy Samples Improves the Inter-observer Agreement of the OLGA Staging System.

PubMed

Cotruta, Bogdan; Gheorghe, Cristian; Iacob, Razvan; Dumbrava, Mona; Radu, Cristina; Bancila, Ion; Becheanu, Gabriel

2017-12-01

Evaluation of severity and extension of gastric atrophy and intestinal metaplasia is recommended to identify subjects with a high risk for gastric cancer. The inter-observer agreement for the assessment of gastric atrophy is reported to be low. The aim of the study was to evaluate the inter-observer agreement for the assessment of severity and extension of gastric atrophy using oriented and unoriented gastric biopsy samples. Furthermore, the quality of biopsy specimens in oriented and unoriented samples was analyzed. A total of 35 subjects with dyspeptic symptoms addressed for gastrointestinal endoscopy that agreed to enter the study were prospectively enrolled. The OLGA/OLGIM gastric biopsies protocol was used. From each subject two sets of biopsies were obtained (four from the antrum, two oriented and two unoriented, two from the gastric incisure, one oriented and one unoriented, four from the gastric body, two oriented and two unoriented). The orientation of the biopsy samples was completed using nitrocellulose filters (Endokit®, BioOptica, Milan, Italy). The samples were blindly examined by two experienced pathologists. Inter-observer agreement was evaluated using kappa statistic for inter-rater agreement. The quality of histopathology specimens taking into account the identification of lamina propria was analyzed in oriented vs. unoriented samples. The samples with detectable lamina propria mucosae were defined as good quality specimens. Categorical data was analyzed using chi-square test and a two-sided p value <0.05 was considered statistically significant. A total of 350 biopsy samples were analyzed (175 oriented / 175 unoriented). The kappa index values for oriented/unoriented OLGA 0/I/II/III and IV stages have been 0.62/0.13, 0.70/0.20, 0.61/0.06, 0.62/0.46, and 0.77/0.50, respectively. For OLGIM 0/I/II/III stages the kappa index values for oriented/unoriented samples were 0.83/0.83, 0.88/0.89, 0.70/0.88 and 0.83/1, respectively. No case of OLGIM IV stage was found in the present case series. Good quality histopathology specimens were described in 95.43% of the oriented biopsy samples, and in 89.14% of the unoriented biopsy samples, respectively (p=0.0275). The orientation of gastric biopsies specimens improves the inter-observer agreement for the assessment of gastric atrophy.

Bee moth (Galleria mellonella) allergic reactions are caused by several thermolabile antigens.

PubMed

Villalta, D; Martelli, P; Mistrello, G; Roncarolo, D; Zanoni, D

2004-09-01

Exposure and contact with bee moth (Galleria mellonella) larvae (Gm) can cause an allergic reaction both in anglers and breeders. We described the case of an amateur fisherman who experienced an allergic reaction using Gm but not using heat-treated Gm (h-Gm) (mummies). The aim of this study was to demonstrate by immunoblotting and radioallergosorbent test (RAST)-inhibition experiments the loss of allergenic epitopes in h-Gm extracts. Galleria mellonella larvae and h-Gm were homogenized and extracted at 10% (w/v) in 0.5 M phosphate-buffered saline, pH 7.4 containing 0.5% NaN(3) for 16 h at 4 degrees C. Gm and h-Gm extracts were electrophoresed in a 10% polyacrylamide precast Nupage Bis-Tris gel at 180 mA for 1 h and the resolved proteins stained with 0.1% Coomassie brilliant blue and the molecular weight calculated. For the immunoblotting detection of allergenic components the resolved extracts were transferred onto a nitrocellulose membrane and incubated with the patient's serum. Bound specific-IgE was detected by peroxidase-conjugated anti-human IgE. RAST inhibition experiments were performed according to the Ceska method. The protein profile of Gm and h-Gm extracts resulted markedly different in number, intensity and the position of bands, indicating that heat-treatment modifies the chemical-physical characteristics of the protein contents. The Gm extract showed a strong-coloured band at 73 kDa and more than 20 components ranging from 12 to 133 kDa; h-Gm showed two main band at 77 and 38 kDa and about 15 faint bands between 20 and 133 kDa apparently without any correspondence to the bands present in the Gm extract. Immunoblotting with the patient's serum demonstrated several bands of reactivity with the Gm extract ranging from 20 to 100 kDa and no recognizable bands, but only a diffuse smear with h-Gm. When used in a RAST inhibition experiment the h-Gm extract demonstrated an inability to compete with the Gm one for the binding to patient's IgE serum. The h-Gm seems to lose the allergenic epitopes and has two advantages for anglers: to avoid new possible sensitizations as well as allergic symptoms in sensitized people, without interfering with their skills and satisfaction in their fishing performance.

Identity of the xerophilic species Aspergillus penicillioides: Integrated analysis of the genotypic and phenotypic characters.

PubMed

Tamura, Miki; Kawasaki, Hiroko; Sugiyama, Junta

1999-02-01

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.

Dual effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109

PubMed Central

Wang, Hong-Mei; Zheng, Nai-Gang; Wu, Jing-Lan; Gong, Cui-Cui; Wang, Yi-Ling

2005-01-01

AIM: To investigate the effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109, and the related gene expression. METHODS: The cultured Eca-109 cells were divided into four groups: E1 group (co-cultured with 8-Br-cAMP for 24 h); E2 group (co-cultured with 8-Br-cAMP for 48 h); C1 group (treated without 8-Br-cAMP for 24 h); and C2 group (treated without 8-Br-cAMP for 48 h). The same concentration of cell suspension of each group was dropped separately onto the slides and nitrocellulose membranes (NCM). The biotin-labeled cDNA probes for c-myc, wild-type (wt) p53, bcl-2 and iNOS were prepared for in situ hybridization. The expressions of epidermal growth factor receptor (EGFR), p38 kinase, FAS, FasL and caspase-3 were detected using immunocytochemistry, and the NOS activity and the ratio of differentiated cells/proliferating cells were examined by cytochemistry. Immunocytochemistry, cytochemistry, and in situ hybridization were separately carried out on both slides and NCM specimens for each group. In addition, TUNEL was used to detect the cell apoptosis rate in each group. RESULTS: The apoptotic rate of E2 group was significantly higher compared to E1 group, while there was no difference in the ratio of differentiated cells/proliferating cells between E1 and E2 groups. The signals of wt p53 and iNOS were markedly stronger, while the signals of c-myc and EGFR were obviously weaker in E1 group than those in C1 group (P<0.05). Moreover, the signals of wt p53, iNOS, p38 kinase, caspase-3 and NOS activity were significantly stronger, whereas, the signals of bcl-2, c-myc and Fas/FasL were markedly weaker in E2 group than those in C2 group (P<0.05). CONCLUSION: The differentiation and apoptosis of human esophageal cancer cell Eca-109 can be induced after 24- and 48-h treatment with 8-Br-cAMP, respectively. Upregulation of wt p53, iNOS and downregulation of c-myc may be associated with differentiation and apoptosis of Eca-109 cells. Furthermore, upregulation of FasL, p38 kinase and caspase-3 as well as downregulation of bcl-2, and Fas may be involved in the apoptosis of Eca-109 cells. PMID:16425431

High resolution as a key feature to perform accurate ELISPOT measurements using Zeiss KS ELISPOT readers.

PubMed

Malkusch, Wolf

2005-01-01

The enzyme-linked immunospot (ELISPOT) assay was originally developed for the detection of individual antibody secreting B-cells. Since then, the method has been improved, and ELISPOT is used for the determination of the production of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, or various interleukins (IL)-4, IL-5. ELISPOT measurements are performed in 96-well plates with nitrocellulose membranes either visually or by means of image analysis. Image analysis offers various procedures to overcome variable background intensity problems and separate true from false spots. ELISPOT readers offer a complete solution for precise and automatic evaluation of ELISPOT assays. Number, size, and intensity of each single spot can be determined, printed, or saved for further statistical evaluation. Cytokine spots are always round, but because of floating edges with the background, they have a nonsmooth borderline. Resolution is a key feature for a precise detection of ELISPOT. In standard applications shape and edge steepness are essential parameters in addition to size and color for an accurate spot recognition. These parameters need a minimum spot diameter of 6 pixels. Collecting one single image per well with a standard color camera with 750 x 560 pixels will result in a resolution much too low to get all of the spots in a specimen. IFN-gamma spots may have only 25 microm diameters, and TNF-alpha spots just 15 microm. A 750 x 560 pixel image of a 6-mm well has a pixel size of 12 microm, resulting in only 1 or 2 pixel for a spot. Using a precise microscope optic in combination with a high resolution (1300 x 1030 pixel) integrating digital color camera, and at least 2 x 2 images per well will result in a pixel size of 2.5 microm and, as a minimum, 6 pixel diameter per spot. New approaches try to detect two cytokines per cell at the same time (i.e., IFN-gamma and IL-5). Standard staining procedures produce brownish spots (horseradish peroxidase) and blue spots (alkaline phosphatase). Problems may occur with color overlaps from cells producing both cytokines, resulting in violet spots. The latest experiments therefore try to use fluorescence labels as a marker. Fluorescein isothiocyanate results in green spots and Rhodamine in red spots. Cells producing both cytokines appear yellow. These colors can be separated much easier than the violet, red, and blue, especially using a high resolution.

Development of solid - based paper strips for rapid diagnosis of Pseudorabies infection.

PubMed

Joon Tam, Yew; Mohd Lila, Mohd Azmi; Bahaman, Abdul Rani

2004-12-01

Pseudorabies (Aujeszky's disease) is an economically significant disease of swine known to cause central nervous disorders, respiratory disease, reproductive failure and mortality in infected pigs. In attempts to eradicate the disease from becoming endemic, early detection is important to prevent further economic losses and to allow for detection and removal of infected pigs in domestic herds. Thus, a rapid and sensitive technique is necessary for the detection of the virus. For rapid and simple examination, an immuno - chromatographic lateral - flow assay system based on immunologic recognition of specific pseudorabies virus antigen was developed by utilising, as signal generator, colloidal gold conjugated to secondary antibody to detect primary or sample antibody in the sera of pseudorabies infected animals. The pseudorabies virus used as a capture antigen in the test strip was first cultivated in VERO cell culture and then purified by sucrose gradient separation to produce the viral protein concentration of 3.8 mg/ml. The standard pseudorabies antigens reacted well with the hyperimmune serum (HIS). The antibody detection system is basically composed of colloidal gold - labelled antibodies fixed on a conjugate pad, and the complementary pseudorabies antigen immobilised onto a nitrocellulose membrane forming capture zone. If the target antibody is present in a specimen, the colloidal gold-labelled antibody will form a complex with the antibody sample. Subsequently, the formed complex will migrate to the capture zone and is then bound to the solid phase via antigen - antibody interaction. As a result, a signal marker is generated by the accumulation of colloidal gold for detection confirmation. The results obtained demonstrated that the optimum combination of pseudorabies antigen needed as the capture reagent and gold conjugate as secondary antibody recognition marker was at a concentration of 0.38mg/ml and at 1:10 dilution factor respectively. The sensitivity of the solid - based test strip towards pseudorabies antibodies was high with a detection limit of 1 to 10,000 - dilution factor. The specificity of the assay was 100% with no cross - reaction being observed with other sera or antibodies. Accurate reading time needed for confirmation of the assay can be completed in 5 min with a whole blood sample of 25 microl. The colloidal gold - labelled antibody is stable at room temperature for 6 months or more (data not shown). Findings from this study indicated that the solid - based test strip assay system provided high sensitivity and specificity for the detection of pseudorabies at low levels of antibody concentration. The assay was rapid, simple, cheap, and does not require any sophisticated equipment. Thus, the solid based test strip will be a useful serological screening technique or for rapid diagnosis of an infectious disease in target populations of animals characterised by heterogeneous antibody responses.

Part I. Mechanisms of injury associated with extracorporeal shock wave lithotripsy; Part II. Exsolution of volatiles

NASA Astrophysics Data System (ADS)

Howard, Danny Dwayne

Part I - Shock waves are focused in extracorporeal shock wave lithotripsy (ESWL) machines to strengths sufficient to fracture kidney stones. Substantial side effects-most of them acute-have resulted from this procedure, including injury to soft tissue. The focusing of shock waves through various layers of tissue is a complex process which stimulates many bio-mechano-chemical responses.This thesis presents results of an in vitro study of the initial mechanical stimulus. Planar nitrocellulose membranes of order 10 um thick were used as models of thin tissue structures. Two modes of failure were recorded: Failure due to cavitation collapsing on or near the membranes, and failure induced by altering the structure of shock waves. Tests were done in water at and around F2 to characterize the extent of cavitation damage, and was found to be confined within the focal region, 1.2 cm along the axis of focus.Scattering media were used to simulate the effects of acoustic nonuniformity of tissue and to alter the structure of focusing shock waves. 40 um diameter (average) hollow glass spheres were added to ethylene glycol, glycerine and castor oil to vary the properties of the scattering media. Multiple layer samples of various types of phantom tissue were tested in degassed castor oil to gauge the validity of the scattering media. The scattering media and tissue samples increased the rise time decreased strain rate in a similar fashion. Membranes were damaged by the decreased strain rate and accumulated effects of the altered structure: After about 20 or so shocks immersed in the scattering media and after about 100 shocks behind the tissue samples. The mode of failure was tearing with multiple tears in some cases from about .1 cm to about 3 cm depending of the number of shocks and membrane thickness.Part II - This work examines the exsolution of volatiles-carbon dioxide from water-in a cylindrical test cell under different pressure conditions. Water was supersaturated with carbon dioxide under various pressures (620 to 1062 kPa), and depressurized rapidly to investigate how carbon dioxide is undissolved, exsolution, and its effects on the surrounding environment. Cavities grow as a result of convective diffusion: They move before depleting carbon dioxide in a given region. The radius of a cavity in this environment grows at a faster rate [...] than that of a cavity at rest [...]. Bubble growth rates were inferred by measuring the bulk liquid using high speed motion pictures. Water in the test-cell is accelerated as a result of buoyancy induced by cavity growth. Cavities are elliptical in shape and grow until mutual interaction causes them to fragment. Accelerations range from 10 to 100 g were measured with velocities ranging from 7 to 13 m/s.

Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis.

PubMed

Rastawicki, Waldemar; Smietafiska, Karolina; Chrost, Anna; Wolkowicz, Tomasz; Rokosz-Chudziak, Natalia

Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections-The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, non- specific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. Recombinant YopD, YopB, YopE and V-Ag proteins of Y enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage ofpositive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA, IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to the protein V-Ag were observed in 32.4%, protein YopB in 27.0% and for the protein YopE in 18.9% serum samples. Immunoglobulin A, and M for the recombinant proteins were found much less frequently than IgG antibodies (respectively 12.2% and 10.8% for V protein-Ag in 10.8% and 14.9% protein and_YopB 2,7% and 10.8% for the protein YopE). Further studies showed that results obtained in recom-dot assay with recombinant protein YopD were comparable with the results of commercial western-blot Yersinia. The study showed that in-house obtained recombinant proteins can be used as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. The most useful was the protein YopD.

The viability and performance characterization of nano scale energetic materials on a semiconductor bridge (SCB)

NASA Astrophysics Data System (ADS)

Strohm, Gianna Sophia

The move from conventional energetic composites to nano scale energetic mixtures (nano energetics) has shown dramatic improvement in energy release rate and sensitivity to ignition. A possible application of nano energetics is on a semiconductor bridge (SCB). An SCB typically requires a tenth of the energy input as compared to a bridge wire design with the same no-fire and is capable of igniting in tens of microseconds. For very low energy applications, SCBs can be manufactured to extremely small sizes and it is necessary to find materials with particle sizes that are even smaller to function. Reactive particles of comparable size to the bridge can lead to problems with ignition reliability for small bridges. Nano-energetic composites and the use of SCBs have been significantly studied individually, however, the process of combining nano energetics with an SCB has not been investigated extensively and is the focus of this work. Goals of this study are to determine if nano energetics can be used with SCBs to further reduce the minimum energy required and improve reliability. The performance of nano-scale aluminum (nAl) and bismuth oxide (Bi2O3) with nitrocellulose (NC), Fluorel(TM) FC 2175 (chemically equivalent to VitonRTM) and Glycidyl Azide Polymer (GAP) as binders where quantified initially using the SenTest(TM) algorithm at three weight fractions (5, 7, and 9%) of binder. The threshold energy was calculated and compared to previous data using conventional materials such as zirconium potassium chlorate (ZPC), mercuric 5-Nitrotetrazol (DXN-1) and titanium sub-hydride potassium per-chlorate (TSPP). It was found that even though there where only slight differences in performance between the binders with nAl/Bi2O 3 at any of the three binder weight fractions, the results show that these nano energetic materials require about half of the threshold energy compared to conventional materials using an SCB with an 84x42 mum bridge. Binder limit testing was conducted to find the critical limit of binder when the output of the SCB declines. The binder was evaluated at 13, 17 and 20% and it was found that the limit amount of binder falls between 17 and 20% by weight of material. Scaling of the SCB bridge was evaluated using a 36x15 mum bridge size and tested using 5, 7 and 9% nAl/Bi2O 3 FC 2175 slurry, creating a functioning SCB compared to previous no-ignition results using TSPP. It was also postulated that the compaction of a secondary material onto the SCB would alter the SCB output during testing. It was found that increased energy values where required for both the 5 and 7% binder amounts and no change was seen at the 9% level.

Polyphasic approach to characterize heterotrophic bacteria of biofilms and patina on walls of the Suburban Bath of the Herculaneum's archaeological excavations in Italy

NASA Astrophysics Data System (ADS)

Ventorino, V.; Pepe, O.; Sannino, L.; Blaiotta, G.; Palomba, S.

2012-04-01

Built between the walls of Herculaneum excavations, one of the world's most important archaeological sites, and the sea in the early 1st cent. AD, the Suburban Bath is one of the best thermal complexes better preserved in ancient times. The entrance opens onto a large courtyard that leads into a hallway well lit by a skylight, impluvium, with a portrait of "Apollo". From this room you can access various parts of the thermae, all beautifully preserved. A single room, mostly occupied by the pool, serving both apodyterium (dressing room) that frigidarium. Among tepidarium and frigidarium there's a room elegantly decorated with stucco and marble. The vestibule opens to the right, through a corridor, onto a waiting room with a floor in signinum opus and into a praefurnium (oven for heating). A large pool of tepidarium, connected with laconicum, a small circular room for the baths sweat, is also present. The calidarium, as usual, has a small tank for hot water and a basin for washing in cold water. Behind the calidarium is the praefurnium, an environment with the boiler for heating the bath. Although the suburban baths are well preserved, unfortunately in you can observe the development of visible microbial coatings. During the biodeterioration process, secondary colonization of wall is due to heterotrophic bacteria and fungi that induce deterioration cause structural as well as aesthetic damage such as the discoloration of materials, the formation of crusts on surfaces and the loss of material. This investigation was carried out sampling the surfaces of walls of different rooms in the Suburban Thermae according to Italian legal procedures. Depending on the samples typology, sampling was carry out using sterile nitrocellulose membranes pressed on the surface of the walls, sterile swabs or with sterile tweezers by tearing out surface material. The samples were suspended in physiological solution and immediately refrigerated until analysis. Isolated colonies grown on PCA plates were purified in the same growth medium by streaking and differentiated by assessing their morphological (phase-contrast microscopy) and biochemical characteristics (Gram-stains KOH-lysis and catalase activity). Cultural-based method allow us to identify by 16S and 26S rRNA partial sequence analysis, heterotrophic bacteria belonging to different genera as Bacillus, Pseudomonas, Aeromonas and Microbacterium. By using this approach, Bacillus-related species (B. benzoevorans, B. megaterium and B. pumilis and B. megaterium/B. simplex group) as well as Aeromonas sobria/Aeromonas salmonicida/Aeromonas hydrophila group, Pseudomonas plecoglossicida and Microbacterium esteraromaticum were isolated in different sample points analysed. DGGE analysis of PCR amplified V3 region of rDNA from DNA directly recovered from samples of biofilms and patina, enabled identification of bacterial species not found using culturable technology, as those closest related to Aeromonas, Paenibacillus, Brevibacterium, Exiguobacterium, Microbacterium, Brevibacterium, Stenothophomonas and Streptomyces. Combination of culture-dependent and independent methods provide a better characterization of heterotrophic microbiota that colonize the surface of ancient decorated walls and can contribute to understand the potential of biodeterioration activity by heterotrophic microorganisms.

What Have We Learned From Decades of CRT, And Where Do We Go From Here?

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

Burnham, A K; Souers, P C; Gagliardi, F J

2006-09-11

The Chemical Reactivity Test, or CRT, has been the workhorse for determining short-to-medium term compatibility and thermal stability for energetic materials since the mid 1960s. The concept behind the CRT is quite simple. 0.25 g of material is heated in a 17 cm{sup 3} vessel for 22 hours at 80, 100, or 120 C, and the yield of gaseous products are analyzed by gas chromatography to determine its thermal stability. The instrumentation is shown in Figure 1, and the vessel configuration is shown in Figure 2. For compatibility purposes, two materials, normally 0.25 g of each, are analyzed as amore » mixture. Recently, data from the past 4 decades have been compiled in an Excel spreadsheet and inspected for reliability and internal consistency. The resulting processed data will be added this year to the LLNL HE Reference Guide. Also recently, we have begun to assess the suitability of the CRT to answer new compatibility issues, especially in view of more modern instrumentation now available commercially. One issue that needs to be addressed is the definition of thermal stability and compatibility from the CRT. Prokosch and Garcia (and the associated MIL-STD-1751A) state that the criterion for thermal stability is a gas yield of less than 4 cm{sup 3}/g for a single material for 22 hours at 120 C. The gases from energetic materials of interest ordinarily have an average molecular weight of about 36 g/mol, so this represents decomposition of 0.5-1.0% of the sample. This is a reasonable value, and a relatively unstable energetic material such as PETN has no problem passing. PBX 9404, which yields 1.5 to 2.0 cm{sup 3}/g historically, is used as a periodic check standard. This is interesting in itself, since the nitrocellulose in the 9404 is unstable and probably has partially decomposed over the decades. However, it is not clear whether this aging of the standard would lead to more or less gas, since the initial gaseous degradation products are captured by the DPA stabilizer. Clearly this is an issue that needs reconsideration. The criterion for compatibility is less clearly correct. Although some LLNL reports say that generation of gas in excess of the materials by themselves is an indication of incompatibility, LLNL reports invariably say that materials are compatible if they generate less than 1 cm{sup 3}/g of gas. There are two problems with this criterion. First, it is not stated whether the gas yield is per gram of energetic material or mixture. Second, a material that generates >2 cm{sup 3}/g by itself could never pass the compatibility tests as stated, because even a mixture of equal masses of that material with a completely inert material would generate >1 cm{sup 3}/g of gas per mixture. Furthermore, Prokosch states that a yield equal to or less than from the materials individually means that no reaction has occurred. Clearly, less gas can not be generated unless some type of interaction has occurred. An obvious example would be mixing CaO with a CO{sub 2}-generating energetic material. In the absence of any direct action of the CaO on the energetic material, the CO{sub 2} product would be captured by the CaO, thereby decreasing the gas yield and liberating considerable heat. In a large, closed volume, this could tip the balance to thermal runaway.« less

Modeling and numerical simulation of interior ballistic processes in a 120mm mortar system

NASA Astrophysics Data System (ADS)

Acharya, Ragini

Numerical Simulation of interior ballistic processes in gun and mortar systems is a very difficult and interesting problem. The mathematical model for the physical processes in the mortar systems consists of a system of non-linear coupled partial differential equations, which also contain non-homogeneity in form of the source terms. This work includes the development of a three-dimensional mortar interior ballistic (3D-MIB) code for a 120mm mortar system and its stage-wise validation with multiple sets of experimental data. The 120mm mortar system consists of a flash tube contained within an ignition cartridge, tail-boom, fin region, charge increments containing granular propellants, and a projectile payload. The ignition cartridge discharges hot gas-phase products and unburned granular propellants into the mortar tube through vent-holes on its surface. In view of the complexity of interior ballistic processes in the mortar propulsion system, the overall problem was solved in a modular fashion, i.e., simulating each physical component of the mortar propulsion system separately. These modules were coupled together with appropriate initial and boundary conditions. The ignition cartridge and mortar tube contain nitrocellulose-based ball propellants. Therefore, the gas dynamical processes in the 120mm mortar system are two-phase, which were simulated by considering both phases as an interpenetrating continuum. Mass and energy fluxes from the flash tube into the granular bed of ignition cartridge were determined from a semi-empirical technique. For the tail-boom section, a transient one-dimensional two-phase compressible flow solver based on method of characteristics was developed. The mathematical model for the interior ballistic processes in the mortar tube posed an initial value problem with discontinuous initial conditions with the characteristics of the Riemann problem due to the discontinuity of the initial conditions. Therefore, the mortar tube model was solved by using a high-resolution Godunov-type shock-capturing approach was used where the discretization is done directly on the integral formulation of the conservation laws. A linearized approximate Riemann Solver was modified in this work for the two-phase flows to compute fully non-linear wave interactions and to directly provide upwinding properties in the scheme. An entropy fix based on Harten-Heyman method was used with van Leer flux limiter for total variation diminishing. The three dimensional effects were simulated by incorporating an unsplit multi-dimensional wave propagation method, which accounted for discontinuities traveling in both normal and oblique coordinate directions. For each component, the predicted pressure-time traces showed significant pressure wave phenomena, which closely simulated the measured pressure-time traces obtained at PSU. The pressure-time traces at the breech-end of the mortar tube were obtained at Aberdeen Test Center with 0, 2, and 4 charge increments. The 3D-MIB code was also used to simulate the effect of flash tube vent-hole pattern on the pressure-wave phenomenon in the ignition cartridge. A comparison of the pressure difference between primer-end and projectile-end locations of the original and modified ignition cartridges with each other showed that the early-phase pressure-wave phenomenon can be significantly reduced with the modified pattern. The flow property distributions predicted by the 3D-MIB for 0, 2, and 4 charge increment cases as well the projectile dynamics predictions provided adequate validation of theory by experiments.

Microbial source tracking and transfer hydrodynamics in rural catchments.

NASA Astrophysics Data System (ADS)

Murphy, Sinead; Bhreathnach, Niamh; O'Flaherty, Vincent; Jordan, Philip; Wuertz, Stefan

2013-04-01

In Ireland, bacterial pathogens from continual point source pollution and intermittent pollution from diffuse sources can impact both drinking water supplies and recreational waters. This poses a serious public health threat. Observing and establishing the source of faecal pollution is imperative for the protection of water quality and human health. Traditional culture methods to detect such pollution via faecal indicator bacteria have been widely utilised but do not decipher the source of pollution. To combat this, microbial source tracking, an important emerging molecular tool, is applied to detect host-specific markers in faecally contaminated waters. The aim of this study is to target ruminant and human-specific faecal Bacteroidales and Bacteroides 16S rRNA genes within rural river catchments in Ireland and investigate hydrological transfer dependencies. During storm events and non-storm periods, 1L untreated water samples, taken every 2 hours over a 48-hour time period at the spring (Cregduff) or outlet (Dunleer), and large (5-20L) untreated water samples were collected from two catchment sites. Cregduff is a spring emergence under a grassland karst landscape in Co. Mayo (west coast of Ireland) and Dunleer is a mixed landuse over till soils in Co. Louth (east coast). From a risk assessment point of view, the catchments are very different. Samples were filtered through 0.2µm nitrocellulose filters to concentrate bacterial cells which then underwent chemical extraction of total nucleic acids. Animal and human stool samples were also collected from the catchments to determine assay sensitivity and specificity following nucleic acid extraction. Aquifer response to seasonal events was assessed by monitoring coliforms and E. coli occurrence using the IDEXX Colisure® Quanti Tray®/2000 system in conjunction with chemical and hydrological parameters. Autoanalysers deployed at each catchment monitor multiple water parameters every 10 min such as phosphorus, nitrogen (nitrate), turbidity, conductivity and flow rate. InStat V 3.06 was used to determine correlations between chemical and microbial parameters (P< 0.05 considered significant).There was a positive correlation between E. coli and phosphorus in Cregduff during rain events (p=0.040) & significant correlation for a non-rain periods (<0.001). There was a positive correlation between E. coli and turbidity in Dunleer during rain events (p=0.0008) and in Cregduff during non-rain periods (p=0.0241). The water samples from Dunleer have a higher concentration of phosphorus than in Cregduff. Host specific primers BacCow-UCD, BacHum-UCD, BacUni-UCD and BoBac were then assayed against both faecal and water extracts and quantified using PCR. BacUni-UCD, BacCow-UCD and BoBac detected faecal contamination in three of the four sample sites in Dunleer and BacHum-UCD detected faecal contamination in one of the sites. The concentrations of the BacUni-UCD qPCR assay were higher in the water samples taken from Dunleer outlet than those taken from Cregduff spring. BacCow-UCD and BacHum-UCD qPCR detected low and very low concentrations, respectively, in water from the Dunleer outlet. The concentrations can be seen changing over the hydrograph event. None of the host-specific assays detected pollution in Cregduff. From the results, it can be seen that Dunleer is more subject to contamination than Cregduff.

Combustion characteristics of SMX and SMX based propellants

NASA Astrophysics Data System (ADS)

Reese, David A.

This work investigates the combustion of the new solid nitrate ester 2,3-hydroxymethyl-2,3-dinitro-1,4-butanediol tetranitrate (SMX, C6H 8N6O16). SMX was synthesized for the first time in 2008. It has a melting point of 85 °C and oxygen balance of 0% to CO 2, allowing it to be used as an energetic additive or oxidizer in solid propellants. In addition to its neat combustion characteristics, this work also explores the use of SMX as a potential replacement for nitroglycerin (NG) in double base gun propellants and as a replacement for ammonium perchlorate in composite rocket propellants. The physical properties, sensitivity characteristics, and combustion behaviors of neat SMX were investigated. Its combustion is stable at pressures of up to at least 27.5 MPa (n = 0.81). The observed flame structure is nearly identical to that of other double base propellant ingredients, with a primary flame attached at the surface, a thick isothermal dark zone, and a luminous secondary flame wherein final recombination reactions occur. As a result, the burning rate and primary flame structure can be modeled using existing one-dimensional steady state techniques. A zero gas-phase activation energy approximation results in a good fit between modeled and observed behavior. Additionally, SMX was considered as a replacement for nitroglycerin in a double base propellant. Thermochemical calculations indicate improved performance when compared with the common double base propellant JA2 at SMX loadings above 40 wt-%. Also, since SMX is a room temperature solid, migration may be avoided. Like other nitrate esters, SMX is susceptible to decomposition over long-term storage due to the presence of excess acid in the crystals; the addition of stabilizers (e.g., derivatives of urea) during synthesis should be sufficient to prevent this. the addition of Both unplasticized and plasticized propellants were formulated. Thermal analysis of unplasticized propellant showed a distinct melt-recrystallization curve, which indicates that a solid phase solution is being formed between SMX and NC, and that SMX would not act as plasticizer. Analysis of propellant prepared with diethyleneglycol dinitrate (DEGDN) plasticizer indicates that the SMX is likely dissolved in the DEGDN. The plasticized material also showed similar hardness and modulus to JA2. However, both plasticized and unplasticized propellants exhibited deconsolidated burning at elevated pressures due to the high modulus of the propellant. Increased amounts of plasticizer or improved processing of the nitrocellulose should be investigated to remedy this issue. Safety characterization showed that sensitivity of the plasticized propellant is similar to JA2. In short, replacing NG with SMX results in a new family of propellants with acceptable safety characteristics and which may also offer improved theoretical performance. Finally, composite propellants based on SMX were theoretically and experimentally examined and compared to formulations based on ammonium perchlorate (AP). Thermochemical equilibrium calculations show that aluminized SMX-based formulations can achieve theoretical sea level specific impulse values upwards of 260 s-- slightly lower than an AP-based composite. Both ignition sensitivity (tested via drop weight impact, electro-static discharge, and BAM friction) and physical properties (hardness and thermal properties) are comparable to those of the AP-based formulations. However, the SMX-based formulation could be detonated using a high explosive donor charge in contact with the propellant, as do other low smoke propellants. Differential scanning calorimetry of the SMX-based propellant indicated an exotherm onset of 140 °C, which corresponds to the known decomposition temperature of SMX. The propellant has a high burning rate of 1.57 cm/s at 6.89 MPa, with a pressure exponent of 0.85. This high pressure sensitivity might be addressed using various energetic and/or stabilizing additives. With high density and performance, smokeless combustion products, and stable combustion, SMX appears to be a viable replacement for existing energetic ingredients in a wide variety of propellant, explosive, and pyrotechnic applications.

Dopamine transporter and vesicular monoamine transporter knockout mice : implications for Parkinson's disease.

PubMed

Miller, G W; Wang, Y M; Gainetdinov, R R; Caron, M G

2001-01-01

One of the most valuable methods for understanding the function of a particular protein is the generation of animals that have had the gene encoding for the protein of interest disrupted, commonly known as a "quo;knockout"quo; or null mutant. By incorporating a sequence of DNA (typically encoding antibiotic resistance to aid in the selection of the mutant gene) into embryonic stem cells by homologous recombination, the normal transcription of the gene is effectively blocked (Fig. 1). Since a particular protein is encoded by two copies of a gene, it is necessary to have the gene on both alleles "quo;knocked out."quo; This is performed by cross-breeding animals with one affected allele (heterozygote) to generate offspring that have inherited two mutant alleles (homozygote). This procedure has been used to generate animals lacking either the plasma membrane dopamine transporter (DAT; Fig. 2) or the vesicular monoamine transporter (VMAT2; Fig. 3). Both DAT and VMAT2 are essential for dopamine homeostasis and are thought to participate in the pathogenesis of Parkinson's disease (1-5). Fig. 1. Maps of the targeting vector and the mock construct. The mouse genomic fragment (clone 11) was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5' end of rat cDNA. The restriction site abbreviations are as follows: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence from transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3' fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the Neo cassette was generated as a positive control. pPNT and pGEM4Z were used to construct knockout and mock vectors, respectively. (Reproduced with permission from ref. 1). Fig. 2. DAT and VMAT2 expression in wild-type and DAT knockout midbrain. DAT immunoreactivity in wild-type (A) and DAT knockout midbrain (B). VMAT2 immunoreactivity in wild-type (C) and DAT knockout midbrain (D). Robust immunoreactivity was observed in the ventral tegmental area and substantia nigra pars compacta and reticulata in the wild-type brain. Note absence of DAT immunoreactivity and modest reduction of VMAT2 immunoreactivity in the DAT knockout. Fig. 3. Characterization of VMAT2 gene disruption. (A) Southern blot analysis of mouse genomic DNA. The Southern blot was prepared with 15 μg of genomic DNA per lane and probed with a 1.4-kb 3' external genomic fragment. +/+, wild type littermates; +/-, heterozygote; -/-, homozygote. (B) RT-PCR analysis of mouse brain poly(A)+ RNA. For each reverse transcription assay, 0.5 μg of poly(A)+ RNA was used. Equal volumes of cDNA templates were used for each PCR assay. The PCR primers used flank the neomycin cassette for the purpose of detecting potential readthrough of the neomycin DNA. The heterozygote has a reduced amount of transcripts compared with the wild-type littermate; the homozygote is devoid of VMAT2 transcripts. G3PDH was used as internal control. (C) Western blot analysis of wholebrain synaptic vesicles. Samples (25 μg) of vesicles were solubilized and separated by SDS-PAGE, transferred to nitrocellulose, subjected to Western blot analysis with anti-VMAT2-Ct (top) or anti-a-tubulin (bottom) antibodies, and developed with chemiluminescence. Molecular mass markers (kDa) are shown to the left. To confirm equal loading and transfer of proteins, the blots were stripped and reprobed with an antibody to α-tubulin. (Reproduced with permission from ref. 1). The importance of DAT in neuronal function is highlighted in animals in which DAT has been genetically deleted (DAT KO) (3). In the homozygote DAT KO mice, released dopamine remains in the extracellular space up to 300 times longer than normal. As expected, these animals display behaviors consistent with persistent activation of dopamine receptors, such as hyperlocomotion. Genetic deletion of VMAT2 reveals the essential role of vesicular storage and release of monoamines. Homozygote VMAT2 knockout mice survive for only a few days, whereas heterozygotes appear normal. Studies performed in homozygote pups and heterozygote adults clearly show that the level of VMAT2 expression calibrates the level of vesicular filling (1,2,bi4). With only 50% of normal VMAT2, heterozygote animals have reduced vesicular filling and release. These alterations in presynaptic monoamine function in the heterozygotes are thought to be responsible for the observed sensitization to the psychostimulants cocaine and amphetamine and to ethanol (1). Knockout animals also appear to parallel the changes that occur in reserpinized animals, suggesting that the adverse actions of this drug are mediated by VMAT2.