Bead Loading Research Articles

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Isotope ratio analysis of actinides, fission products, and geolocators by high-efficiency multi-collector thermal ionization mass spectrometry

A ThermoFisher “Triton” multi-collector thermal ionization mass spectrometer (MC-TIMS) was evaluated for trace and ultra-trace level isotope ratio analysis of actinides (uranium, plutonium, and americium), fission products and geolocators (strontium, cesium, and neodymium). Total efficiencies (atoms loaded to ions detected) of up to 0.5–2% for U, Pu, and Am, and 1–30% for Sr, Cs, and Nd can be reported employing resin bead load techniques onto flat ribbon Re filaments or resin beads loaded into a millimeter-sized cavity drilled into a Re rod. This results in detection limits of <0.1 fg (10 4 atoms to 10 5 atoms) for 239–242+244Pu, 233+236U, 241–243Am, 89,90Sr, and 134,135,137Cs, and ≤1 pg for natural Nd isotopes (limited by the chemical processing blank) using a secondary electron multiplier (SEM) or multiple-ion counters (MICs). Relative standard deviations (RSD) as small as 0.1% and abundance sensitivities of 1 × 10 6 or better using a SEM are reported here. Precisions of RSD ≈0.01–0.001% using a multi-collector Faraday cup array can be achieved at sub-nanogram concentrations for strontium and neodymium and are suitable to gain crucial geolocation information. The analytical protocols reported herein are of particular value for nuclear forensic and nuclear safeguard applications.

Biodegradation of coumaphos, chlorferon, and diethylthiophosphate using bacteria immobilized in Ca-alginate gel beads

Calcium-alginate immobilized cell systems were developed for the detoxification and biodegradation of coumaphos, an organophosphate insecticide, and its hydrolysis products, chlorferon and diethlythiophosphate (DETP). Optimum bead loadings for bioreactor operation were found to be 200 g-beads/L for chlorferon degradation and 300 g-beads/L for DETP degradation. Using waste cattle dip (UCD) solution as substrate, the degradation rate for an immobilized consortium of chlorferon-degrading bacteria was five times greater than that for freely suspended cells, and hydrolysis of coumaphos by immobilized OPH + Escherichia coli was 2.5 times greater. The enhanced degradation of immobilized cells was due primarily to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of the cells caused by Ca-alginate immobilization may have contributed to increased reaction rates. Degradation rates for repeated operations increased for successive batches indicating that cells became better adapted to the reaction conditions over time.

Naphthalene Mass Transfer from a Non-Aqueous Phase Liquid (NAPL) in Rotating Baffled and Bead Mill Bioreactors

Rotating bead mill and baffled bioreactors have earlier been shown to provide excellent mass transfer and bioremediation rates for naphthalene particulates. In this study, the mass transfer rates of naphthalene and methylnaphthalenes from NAPL into water in both the bead mill and baffled bioreactors are reported. The values of KLa ranged between 1.0 h−1 and 42 h−1, similar to values observed with suspended PAH particulates, increasing with bead loadings up to 50% by volume, bead size up to 5.0 mm, rotation rate up to 50 RPM, oil loading up to 72 mL (7.2% volume fraction) and naphthalene loading up to 1000 mg/L (based on the water phase). Baffled bioreactors provided similar volumetric mass transfer coefficients as bead mill bioreactors, but without the loss of working volume due to the presence of solid beads.

Multifunctionalized Biocompatible Microspheres for Sensing

We present our achievements in the synthesis and application of multifunctionalized, cross-linked, polystyrene microspheres, which remarkably and quite generally are taken up by all cell types studied to date. Importantly, the nature of these synthetic beads allows multistep solid-phase chemistry and the ability to bind essentially any molecule/sensor/nucleic acid to them. These microspheres have a number of advantages over other cellular delivery approaches. First, a diverse range of compounds can be attached to the microspheres, and we have demonstrated that these materials are effectively delivered into cells; second, the cellular cargo can be modulated through modification of bead loading; third, they are large enough to be visualized using standard microscopy techniques; and fourth, their cargos are not diluted within the cell. Populations of cells containing beads can be readily sorted from other cells for subsequent analysis with very high, but controllable, uptake rates, which can be modulated through modulation of the bead size and incubation time. Recent work has focused on the development of these microspheres as sensors for intracellular calcium and pH.

The release of hepatitis B core antigen from Escherichia coli by batch mode bead milling

The performance of a batch model bead mill on the release of hepatitis B core antigen (HBcAg) from Escherichia coli was investigated in this study. The operating parameters examined were impeller tip speed (8–14 m/s), biomass concentration [5–20% (w/v)] and bead loading [65–80% (v/v)]. The highest yield (24.3 mg/g cell) and rate constant (0.471 l/min) of HBcAg release were achieved at impeller tip speed of 14 m/s. However, the high-shear stress under these operating conditions caused damage of the HBcAg. The highest yield (22.7 mg/g cell) and rate constant (0.344 l/min) of HBcAg release were observed at biomass concentration of 20% (w/v). There was no significant effect of bead loading on the performance of bead milling being observed. In conclusion, the optimal operating condition for the release of HBcAg was at bead loading of 75% (v/v), biomass concentration of 20% (w/v) and impeller tip speed of 10 m/s.

Templating Silica Aerogel with Polystyrene to Improve Their Mechanical Properties

The sol-gel methods applied in the synthesis of aerogels lead to the formation of a disordered silica network. The resulting aerogel has poor structural definition that leads to poor mechanical properties. The work presented details our efforts to create a new hierarchical mesoporous silica aerogel. These meso-porous aerogels were formed utilizing a templating technique using polystyrene beads with varying diameters, 50 nm to 2 μm, dispersed during sol-gel polymerization. The resulting gel was super-critically dried creating a silica aerogel templated with polystyrene beads. The polystyrene beads were then thermal oxidized creating meso-porous silica aerogel monolith. The surface area, pore volume, pore diameter, and mechanical properties of the templated aerogels were determined. Interestingly the mechanical properties of the meso-porous aerogel were significantly improved. These improvements appear to be directly related to the polystyrene bead diameter and loading.

Introduction of Caged Peptide/Protein into Cells Using Bead Loading: Figure 1.

INTRODUCTIONActivation and inactivation of proteins using photoactivation of caged peptides or proteins offers insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group via a limited number of critical functional groups in the biomolecules. Under carefully defined conditions, a brief irradiation with UV light (320-400 nm) can lead to an intramolecular photoisomerization reaction that induces the cleavage of the chemical bond between the cage group and the unmodified, biologically active species. Three strategies can be used to introduce caged compounds into cells: cell-permeant peptide vectors, bead loading, and microinjection. In bead loading, cells are bathed in a medium containing the peptide/protein of interest. Glass beads are then sprinkled onto the cells. The beads create temporary holes in the cell membranes, allowing some of the peptide molecules to enter cells at random. Due to the low efficiency of this technique, a large amount of protein or peptide is needed to label a relatively small number of cells. If the peptides or proteins are not fluorescent, it is useful to co-load the cells with an inert fluorescent marker, such as rhodamine dextran, to indicate successful loading. A representative bead-loading protocol using fish keratocytes is given here.

Microreactor Array Assembly, Designed for Diversity Oriented Synthesis Using a Multiple Core Structure Library on Solid Support

AbstractThe application of spatially encoded principles in solid phase combinatorial synthesis requires no chemical or physical coding strategies. The resulting products are encoded by their position inside the array and their synthesis history. The advantages of microreactor arrays for solid phase synthesis as one of the embodiments in the field of microreaction technology are discussed. Here, we review the reactor design, necessary process steps, and a strategy for the diversity oriented array synthesis. In particular, the glass‐made microreactor and its assembly for 1563 parallel solid phase reactions, which can be performed at temperatures up to 120 °C, are described. Bead loading and liquid handling steps were adapted to this array. The production of large libraries demands suitable synthesis protocols and building blocks. The optimization of appropriate synthesis conditions is a time‐consuming process. A multiple core structure library approach for the efficient synthesis of diverse heterocyclic libraries is described. The aim of this work was to prove the feasibility of the glass‐microreaction array for massive parallel library synthesis.

Evaluation of Fatigue Strength Improvement of Ship Structural Details by Weld Toe Grinding

In order to strengthen or repair the welded structural members or fatigue damaged areas, various surface treatment methods such as grinding, shot peening and/or hammer peening are commonly employed among other methods available. While the weld toe grinding method is known to give 3~4 times of fatigue strength improvement, this improvement may significantly vary according to weld bead shapes and loading modes. In this context, a series of fatigue tests is carried out for three types of test specimens that are typically found in ship structures. Weld burr grinding is carried out using an electric grinder in order to remove surface defects and improve weld bead profiles. The test results are compared with the same type of test specimen without applying the fatigue improvement technique in order to obtain a quantitative measure of the fatigue strength improvement. Moreover, structural stress method is employed to evaluate the effectiveness of the method in evaluating the fatigue strength improvement of welded structures.

Chitosan Beads as a New Gastroretentive System of Verapamil

The main objective of this project is to design a new extended release gastroretentive mutiparticulate delivery system for verapamil (VP) by incorporation into hydrogel beads made of chitosan. The beads were formed by dropping solutions of VP and chitosan in a solution of tripolyphosphate using a syringe pump with adjustable constant rate. The formed beads were then further crosslinked using glutraldehyde and the excess glutraldehyde were then washed. The physical properties of the prepared beads such as beads sizes, shapes, friabilities and loading efficiencies were determined. The floating characteristics and the release profiles were also studied. The produced beads from all batches showed a very good spherical geometry with mean diameter in the range of 1.3 – 2.0 mm. The drug loading efficiency was around 42% for all batches. The % friabilities were less than 1% indicating that the beads surfaces are highly resistant to attrition. Batches B3 and B4 (prepared using medium molecular weight chitosan) showed both the slowest release rate among all the prepared batches and showed good floating characteristics comprising short onset (around 5 minutes) and the long duration of buoyancy (more than 6 hours). All the batches exhibited a kinetic model of combined mechanism of diffusion partially through a swollen matrix and partially through water-filled pores. The preparation of chitosan beads using this technique would provide a simple and commercially viable method of preparation of chitosan beads for controlling the release of some drugs.

Kinetics of heavy metal uptake by vegetation immobilized in a polysulfone or polycarbonate polymeric matrix

The ability of four common vegetations – wood, grass, compost, and peat moss – to remove cadmium, chromium, and lead from dilute aqueous solutions is investigated. Dried ground vegetations are immobilized in polysulfone, and poly (bisphenyl A) carbonate to form spherical beads through a phase inversion process. The beads are contacted with a dilute aqueous solution containing metal ions of interest. The removal of metal ions from the solution is monitored over the course of the experiment and the first-order kinetics parameters estimated. The rates of removal as well as the equilibrium bead loadings are shown to be affected by both the choice of vegetation and the choice of polymer.

A Renewable Electrochemical Magnetic Immunosensor Based on Gold Nanoparticle Labels

A particle-based renewable electrochemical magnetic immunosensor was developed by using magnetic beads and gold nanoparticle labels. Anti-IgG antibody-modified magnetic beads were attached to a renewable carbon paste transducer surface by magnet that was fixed inside the sensor. Gold nanoparticle labels were capsulated to the surface of magnetic beads by sandwich immunoassay. Highly sensitive electrochemical stripping analysis offers a simple and fast method to quantify the capatured gold nanoparticle tracers and avoid the use of an enzyme label and substrate. The stripping signal of gold nanoparticles is related to the concentration of target IgG in the sample solution. A transmission electron microscopy image shows that the gold nanoparticles were successfully capsulated to the surface of magnetic beads through sandwich immunoreaction events. The parameters of immunoassay, including the loading of magnetic beads, the amount of gold nanoparticle conjugate, and the immunoreaction time, were optimized. The detection limit of 0.02 microg ml(-1) of IgG was obtained under optimum experimental conditions. Such particle-based electrochemical magnetic immunosensors could be readily used for simultaneous parallel detection of multiple proteins by using multiple inorganic metal nanoparticle tracers and are expected to open new opportunities for disease diagnostics and biosecurity.

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.

Microchip-based electrochromatography: designs and applications

Different techniques and methods of electrochromatography on “lab on a chip” devices are reviewed. Described approaches include open-channel microchip electrochromatography relying on C 8, C 18 and novel gold nanoparticle (GNP) coating of microchannel wall; packed-channel microchip electrochromatography with new ways of automated loading and unloading of conventional octadecylsilica beads; monolith-based microchip electrochromatography with tailored casting of stationary phase at the specific places of microfluidic network and novel photolitographically fabricated collocated monolithic structures. Specific issues related to the microchip electrochromatography, i.e. importance of high aspect ratio of the microchannels in the open-channel electrochromatography or approaches eliminating the wall effect in the monolith-based electrochromatography, are discussed. Various applications for environmental, pharmacological, genomic and proteomic analysis are described. The operation parameters of reviewed microsystems are summarized in easy-to-read tables.

Measurement of the FMOC loading of protected amine-functionalised polymer beads

This paper presents an HPLC based procedure that has been developed for the determination of the 9-fluorenylmethoxycarbonyl (FMOC) content of protected amine-functionalised polymer beads. FMOC reagents are frequently employed both in the protection of amines and in their determination. The procedure utilises the stoichiometric base cleavage of dibenzofulvene from the protected amine using 1,8-diazabicyclo undec-7-ene. This stearically hindered base prevents the adduct formation that occurs with alternative base systems and measurement performance is enhanced by the incorporation of anthracene into the system as a non-reactive internal standard. HPLC separation of the reaction products permits the measurement of FMOC in the presence of additional chromophores that might otherwise impede direct measurement by UV–vis spectrophotometry. The procedure has been evaluated for the measurement of the FMOC content of protected amine-modified polymer beads employed in combinatorial solid phase synthesis.

Polymer beads in microtubule-filled composites: Effects on length distributions and permittivities

The effects of adding poly(methylmethacrylate) beads to tubule-filled composites were examined in terms of changes in tubule distribution and dielectric properties of the composites. The beads had an average diameter of 51±29 μm, whereas the copper-coated tubules, which were shaped like hollow cylinders and derived from a phospholipid template, had an average length of 30±22 μm and an average diameter of 1 μm. Incorporating a small amount of beads increased the permittivity value of the composite, but adding more beads resulted in a dramatic decrease in the permittivity value. In fact, a permittivity maximum appeared at around 5 vol % beads for various tubule loadings. The results could be explained in terms of volume exclusion and decreased tubule length effects. At low bead loadings, the volume exclusion effect dominated as the tubules were pushed closer together, resulting in a higher permittivity value. At higher bead loadings, the tubules were broken into shorter pieces and this became the dominant effect. Consequently, the shorter tubules formed fewer conducting pathways and the permittivity declined in value. In addition, the De Loor model for composites containing a dielectric matrix and conductors as fillers correctly predicted a permittivity maximum and provided the best fit to the permittivity data.

Visualizing telomere dynamics in living mammalian cells using PNA probes.

Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A fluorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to specifically associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital fluorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed significant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.

Modification and control of currents on monopole antennas using magnetic bead loading

Several methods to modify the currents on electrically long monopole antennas are investigated. The radiation pattern, input impedance, and resonant frequency are calculated for several configurations of monopoles loaded with magnetic materials, both as coatings and as small beads. Proper application of such loading is shown to cause the radiated fields and resonant frequency to mimic a shorter quarter-wave monopole.

The mixing of a polymeric powder and the grinding medium in a shaker bead mill

In this work, we have studied the influence of the operating conditions on the mixing of a polymeric powder and the glass beads in a shaker bead mill. The parameters studied are the position of polymer, polymer load, bead load, diameters of beads and polymer, frequency and amplitude of vibration. In the system, three phenomena govern the mixing of the constituents and the homogeneity of the system: downward percolation, upward percolation and diffusion. The rate of each phenomenon depends on the operating conditions. We have noted that the acceleration of vibration is the most important parameter.