Bead Mass Research Articles

IL‐6 adsorption dynamics in hemoadsorption beads studied using confocal laser scanning microscopy

Sepsis is characterized by a systemic inflammatory response caused by infection, and can result in organ failure and death. Removal of inflammatory mediators such as cytokines from the circulating blood is a promising treatment for severe sepsis. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using biocompatible, polymer sorbent beads. In this study, we used confocal laser scanning microscopy (CLSM) to directly examine adsorption dynamics of a cytokine (IL-6) within hemoadsorption beads. Fluorescently labeled IL-6 was incubated with sorbent particles, and CLSM was used to quantify spatial adsorption profiles of IL-6 within the sorbent matrix. IL-6 adsorption was limited to the outer 15 microm of the sorbent particle over a relevant clinical time period, and intraparticle adsorption dynamics was modeled using classical adsorption/diffusion mechanisms. A single model parameter, alpha = q(max) K/D, was estimated by fitting CLSM intensity profiles to our mathematical model, where q(max) and K are Langmuir adsorption isotherm parameters, and D is the effective diffusion coefficient of IL-6 within the sorbent matrix. Given the large diameter of our sorbent beads (450 microm), less than 20% of available sorbent surface area participates in cytokine adsorption. Development of smaller beads may accelerate cytokine adsorption by maximizing available surface area per bead mass.

Substrate and water adsorption phenomena in a gas/solid enzymatic reactor

The adsorption of water and substrates to dry deposited alcohol dehydrogenase from Lactobacillus brevis (LBADH) is studied in a continuous enzymatic gas/solid reactor. This work is aiming at obtaining a deeper and more thorough understanding of the enzyme microenvironment in the gas/solid system of acetophenone reduction with concomitant oxidation of 2-propanol. Extensive water adsorption studies showed that the effect of sucrose in the enzyme preparation on the water adsorption isotherm is significant for water activities exceeding 0.5 and reaches a factor 2 with respect to bead mass at water activity of 0.9. Significant hysteresis during water desorption is identified, resulting in up to 0.6 mg(water)/mg(protein), for lyophilized enzyme preparation and up to 10 mg(water)/mg(protein) for deposited enzyme preparation. The adsorption of the substrates is quantified here for the first time. Whereas the adsorption of the main substrate, acetophenone, may reach a significant level of up to 6 mg/mg(protein), at an acetophenone activity of 0.32, the secondary substrate, 2-propanol is not adsorbed at a detectable degree. The presence of water leads to a decrease of the adsorbed amount of acetophenone, by approximately 25%, at a water activity of 0.54.

Fibrillin Microfibrils: A Key Role for the Interbead Region in Elasticity

Fibrillin microfibrils have essential roles in elastic fiber formation and elastic tissue homeostasis, as well as transforming growth factor-beta sequestration. A role for fibrillin microfibrils in tissue elasticity has been implied by their ability to increase periodicity from 56 to 150 nm. In this study, we found that microfibril periodicity and structure are dependent on the ionic strength of the buffer and Ca(2+) concentration; we then used these properties of the microfibril to trap conformation intermediates. Transmission electron microscopy imaging of microfibrils with a range of periodicities between 56 and 154 nm revealed a gross conformational change in the interbead region that accommodates the length change. At periodicities below 85 nm, four thin filaments are visualized in the interbead region, but at periodicities greater than 85 nm, two thick filaments are seen. The diameter of the bead remains almost constant at all periodicities, but there is a decrease in stain-exclusion above 85 nm periodicity, which is likely to correspond to a decrease in bead mass. Additionally, we identified eight molecules in cross-section through a microfibril, allowing us to understand microfibril organization in three dimensions. In conclusion, when microfibrils extend, there is a large molecular rearrangement within the interbead region, and this highlights a possible role for Ca(2+) in stabilizing the microfibril architecture and moderating extension in vivo.

Flexible Acoustic Particle Manipulation Device with Integrated Optical Waveguide for Enhanced Microbead Assays

Realisation of a device intended for the manipulation and detection of bead-tagged DNA and other bio-molecules is presented. Acoustic radiation forces are used to manipulate polystyrene micro-beads into an optical evanescent field generated by a laser pumped ion-exchanged waveguide. The evanescent field only excites fluorophores brought within approximately 100 nm of the waveguide, allowing the system to differentiate between targets bound to the beads and those unbound and still held in suspension. The radiation forces are generated in a standing-wave chamber that supports multiple acoustic modes, permitting particles to be both attracted to the waveguide surface and also repelled. To provide further control over particle position, a novel method of switching rapidly between different acoustic modes is demonstrated, through which particles are manipulated into an arbitrary position within the chamber. A novel type of assay is presented: a mixture of streptavidin coated and control beads are driven towards a biotin functionalised surface, then a repulsive force is applied, making it possible to determine which beads became bound to the surface. It is shown that the quarter-wave mode can enhance bead to surface interaction, overcoming potential barriers caused by surface charges. It is demonstrated that by measuring the time of flight of a microsphere across the device the bead size can be determined, providing a means of multiplexing the detection, potentially detecting a range of different target molecules, or varying bead mass.

Dissolution of uranium metal without hydride formation or hydrogen gas generation

This study shows that metallic uranium will cleanly dissolve in carbonate-peroxide solution without generation of hydrogen gas or uranium hydride. Metallic uranium shot, 0.5–1 mm diameter, was reacted with ammonium carbonate–hydrogen peroxide solutions ranging in concentration from 0.13 M to 1.0 M carbonate and 0.50 M to 2.0 M peroxide. The dissolution rate was calculated from the reduction in bead mass, and independently by uranium analysis of the solution. The calculated dissolution rate ranged from about 4 × 10 −3 to 8 × 10 −3 mm/h, dependent primarily on the peroxide concentration. Hydrogen analysis of the etched beads showed that no detectable hydrogen was introduced into the uranium metal by the etching process.

Mass and position determination of attached particles on cantilever based mass sensors

An analytical expression relating mass and position of a particle attached on a cantilever to the resulting change in cantilever resonant frequency is derived. Theoretically, the position and mass of the attached particle can be deduced by combining measured resonant frequencies of several bending modes. This finding is verified experimentally using a microscale cantilever with and without an attached gold bead. The resonant frequencies of several bending modes are measured as a function of the bead position. The bead mass and position calculated from the measured resonant frequencies are in good agreement with the expected mass and the position measured.

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as ∼7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate d/l-glyceraldehyde with a Ki of 1.16μM.

Effect of Nuclear Actin on Endothelial Nitric Oxide Synthase Expression

Previously, we showed that the 27nt repeat polymorphism in endothelial nitric oxide synthase (eNOS) intron 4 was associated with altered eNOS mRNA and protein levels, nitric oxide (NO) production and vascular disease risk; the 27-nt repeats had a cis-acting role in eNOS promoter function. In the present study, we investigated nuclear protein that binds the 27nt repeat and mediates eNOS expression. Using 5'-biotin-labeled 27nt DNA duplex and streptavidin-agarose beads pull-down assay and mass spectrometry, we identified that nuclear beta-actin was one of the major 27nt binding proteins. Using the pGL3 reporter vectors containing the 5x27nt repeats as an enhancer in an in vitro transcription assay, we found that exogenous beta-actin significantly increased reporter gene transcription efficiency. The beta-actin's upregulating effect was compromised when exogenous 27nt RNA duplex was added. Furthermore, the eNOS expression was reduced when beta-actin gene was silenced by specific siRNA, and actin overexpression upregulated eNOS expression >3-fold. Our data demonstrate that beta-actin as a transcription factor stimulates eNOS expression; and the transcriptional effect appears to be 27nt-dependent. Our findings represent a novel molecular mechanism regulating eNOS expression, which could potentially lead to discoveries of eNOS specific pharmaceutical agents, eg, active peptides, with clinical applications.

Application of microbead biological filters

The application of floating microbead filters to aquaculture is reviewed and discussed. The microbead filter is distinctly different from the more commonly used floating bead filters that are used today. Conventional bead filters work in pressured vessels and use a media that is only slightly buoyant. The required mass of beads for the volume required make the media a relatively expensive component of a floating bead filter in contrast to sand or microbead media that is much less expensive on a per volume basis. Microbead filters use polystyrene beads (microbead) that are 1–3mm in diameter (floating bead filters use media approximately 3mm in diameter also). Microbead have an overall bulk density of 16kg/m3 and a specific surface area of 3936m2/m3 (for 1mm beads). This material can be obtained commercially in bulk for roughly US$ 4kg−1 of material. Biological filters that use microbeads for their nitrifying substrate can be thought of as a trickling bio-filter in terms of how the flow distribution and collection mechanics are designed and operated. For design purposes, microbead filters can be assumed to nitrify approximately 1.2kg of TAN/m3 of media per day for warm water systems with influent ammonia–nitrogen levels from 2 to 3mg/l. For cool water applications, rates should be assumed to be 50% of warm water rates or use rates similar to those used for fluidized sand beds. Designs and results in several applications are presented. Microbead filters have been used successfully by several commercial growers after being first introduced in the mid 1990s. Effects of capitalization for equipment and buildings upon production costs is discussed and presented in graphical form.

Translating DNA into synthetic molecules.

Translating DNA into synthetic molecules.

THE CAPTURE OF ESCHERICHIA COLI O157:H7 FOR LIGHT ADDRESSABLE POTENTIOMETRIC SENSOR (LAPS) USING TWO DIFFERENT TYPES OF MAGNETIC BEADS1

Two types of commercially available, streptavidin‐coated magnetic beads (SAMB) were labeled with the same biotinylated anti E. coli O157 antibodies. Labeled SAMB and other necessary antibody derivatives needed for Light‐addressable Potentiometric Sensor (LAPS) detection were used to capture E. coli O157:H7 in solution. The LAPS signal intensity was then used to determine the overall bacterial capture efficiency. The static and hydrodynamic properties of the beads were then used to correlate with determined efficiencies. It was found that the capture increased as the total mass of SAMB increased. Also, greater capture with SAMB of a higher density was observed with the same bead mass. In addition, the SAMB of higher density also required less time of rocking to reach a maximum capture. These observations are in agreement with the predictions from the hydrodynamic properties of tested SAMB.

Growth and production of cholesterol oxidase by alginate-immobilized cells of Rhodococcus equi No. 23.

Rhodococcus equi No. 23 was immobilized in calcium alginate. No detrimental effect on the viability of the test organism was observed during the immobilization procedure. Approx. 98% of the cell population originally present in the alginate solution were immobilized in the gel beads. When the cells of an equal volume of the culture, obtained respectively at exponential phase (12 h preculture), late-exponential phase (20 h preculture) or stationary phase (36 h preculture) were immobilized, the gel beads prepared with the stationary-phase culture were found to contain the highest cell population [about 10(8) colony-forming units (CFU)/g of beads]. In addition, gel beads, prepared with late-exponential-phase culture, exhibited the highest production of cholesterol oxidase (CholOx) after 48 h of incubation. Increasing the bead mass from 3.5 to 14.0 g/100 ml of medium increased CholOx production. However, further increasing the bead mass resulted in a reduction of CholOx production. Furthermore, on the basis of a similar initial cell population, the alginate-immobilized cells of R. equi No. 23 produced a significantly higher amount of CholOx (P<0.05) than did the free cells.

撹はん型ビーズミルによるパン酵母内酵素の放出過程

Release process of enzymes from baker's yeast was investigated experimentally by using an agitating bead mill. The enzymes examined were acid phosphatase, alcohol dehydrogenase, alkaline phosphatase, and invertase. The release process of enzymes was affected significantly by operating conditions of the mill, such as the concentration of cell suspension, the agitating speed of impeller, the volume of cell suspension, and the mass of glass beads. During the disruption of baker's yeast, the release of invertase in the cell wall was the fastest, followed by acid phosphatase and alcohol dehydrogenase in the periplasm and cytoplasm, and alkaline phosphatase bound with the cell membrane was the slowest. The release rate of enzymes was directly proportional to the product of the activity of unreleased enzymes and the agitating speed of impeller. Experimental models based on the kinetics were found to describe the release process of enzymes from baker's yeast disrupted by the agitating bead mill.

The attachment and cleavage of phenols from solid supports and their single bead mass spectral analysis

The attachment of simple phenols to chloromethyl polystyrene, and their rapid cleavage at room temperature is described. Further, phenols from single resin beads can be unequivocally identified by Fourier transform ion cyclotron resonance mass spectrometry of the dansylates prepared in situ.

Wet grinding of gibbsite in a bead-mill

The phenomena (breakage, abrasion, chipping, etc.) taking place during wet grinding in a bead-mill have been analyzed using a set of tools developed to allow for a quantitative description of the particle shape, in complement of the classical approach based on selection and breakage functions. Different operation conditions have being examined for the grinding of gibbsite: concentration of the suspension, mass and diameter of grinding beads, type of gibbsite (“radial” and “mosaic”). Large beads favor the initial breakage of “radial” gibbsite particles when chipping is observed with small beads. Abrasion is clearly observed in the case of autogenous grinding. “Mosaic” gibbsites were found to be more prone to abrasion in the early moments of the grinding than “radial” gibbsite.

STEM mass mapping of type VI collagen microfibrils: Implications for chain composition and alternative splicing

Type VI collagen is a major structural interactive extracellular matrix macromolecule which forms double-beaded microfibrils in the extracellular space. These microfibrils can be isolated in native form from collagenase digest of skin fibroblast cell layers by chromatography on Sepharose CL-2B. Scanning transmission electron microscope (STEM) mass mapping showed a periodicity of 104 nm and a mass/bead of 1500 kDa. In addition, there was an uneven mass distribution along the bead. Microfibrils arise by end-to-end aggregation of tetramers which should produce microfibrils with bead mass of ≈2000 kDa and homogeneous mass distribution across the bead. Reduction in the mass across the bead implies either altered chain composition, alternative splicing or proteolytic degradation. The pattern of splicing of type VI chains in human tissues and their biological implications are poorly defined. In this study, we have examined the abundance of alternatively spliced forms of theα2(VI) andα3(VI) chains in human skin fibroblasts. BothαC2 andα2C2a variants, but not theα2C2a′ form, were expressed in these although theα2C2 form appeared more abundant. Theα3(VI) N7 domain was frequently spliced out of transcripts present in skin fibroblasts, whereas theα3(VI) N9 domain was always present. No consistent pattern of splicing was observed and splicing is unlikely to account for the reduced mass observed in type VI collagen microfibrils.

Vibration and wave localization in a nearly periodic beaded string

The dynamics of an exact, linear model of a string with attached beads are examined. A study of the perfectly periodic system is performed using a transfer matrix formulation. A closed-form solution for the natural frequencies of the finite system is obtained. The relationship between natural frequencies and the passbands and stop bands of propagating waves is studied. The effect of random disorder of (a) bead spacing and (b) bead mass is examined and interesting, fundamental differences are observed. In the case of bead-spacing disorder both weak and strong localization occur, whereas bead-mass disorder only causes weak localization. Localization, the spatial confinement of vibration energy due to periodicity breaking disorder, is quantified by the localization factor. Analytical approximations of the localization factor are derived, in the limits of large and small coupling to disorder ratios. The results are verified through Monte Carlo simulations. The natural frequencies and modes of the disordered s...

Production of Concentrated Lactococcus lactis subsp. cremoris Suspensions in Calcium Alginate Beads

The effect of simultaneous modification of medium composition and growth conditions on the production of Lactococcus lactis subsp. cremoris biomass in calcium alginate beads was studied by the response surface method. Statistical methods of data analysis for unbalanced experiments are illustrated. The media tested were whey, whey supplemented with yeast extract and/or meat extract, milk, and the commercial medium Gold Complete (Nordica). Fermentations were performed at 23 degrees C under pH control (5.6, 6.0, 6.4, or 6.8). In one complete series, 1% CaCO(3) was added to the growth media. There were strong interactions between CaCO(3) and media, CaCO(3) and pH level, and CaCO(3), media, and pH level. In media with CaCO(3), all first-order interactions between media, pH, and sampling time were significant. The addition of CaCO(3) increased cell counts in whey-meat extract medium, but no significant difference was found with the other media. Uncoupling between growth and acidification occurred between 16 and 22 h. Highest counts were obtained on milk and Gold Complete (6 x 10/g). In CaCO(3)-containing media, pH influenced cell counts only in whey and in Gold Complete (pH 5.6 and 6.0 giving the best results); pH also influenced the bead mass obtained at the end of the fermentation. Biomass production in alginate gels is proposed as a method of obtaining concentrated cell suspensions without centrifugation or filtration.

An NMR method of measuring the mass of magnetic beads in a low density fluid (biomedical measurement)

A nuclear magnetic resonance (NMR) method of determining the mass of the magnetic particles present in a fluid by observing the relative frequency shift of an NMR signal is proposed. Experimental tests have been carried out to find the ratio of the mass of the beads to the total mass of the liquid with various densities by using this method. The method can be used to measure masses in a fluid containing a low concentration of magnetic particles. >

The effect of size and mass on the film thickness of beads coated in fluidized bed equipment

The relationship between the film thickness on a bead, and the bead's size and mass in a polydispersed system was studied. Beads with a size distribution in the no. 14–20 mesh range were coated using Glatt fluidized bed units equipped with a Wurster insert. The coated beads were separated into narrower size fractions and dissolution testing of each fraction was performed using the USP basket method. The larger beads exhibited much slower release rates compared to the smaller beads, and the differences could not be explained by the relative surface areas. Examination of the beads by scanning electron microscope indicated that the larger or heavier beads received a thicker film compared to the smaller or lighter beads. This trend was attributed to differences in the fluidization patterns and velocities of the various sized beads.