Preparing a highly specific inert immunomolecular-magnetic beads for rapid detection and separation of <Emphasis Type="Italic">S. aureus</Emphasis> and group G <Emphasis Type="Italic">Streptococcus</Emphasis>

The rapid detection and separation of Staphylococcus aureus and group G Streptococcus was based on the affinity chromatography interactions between Fc fragment of human IgG and protein A/G (located on the cell wall of S. aureus and group G Streptococcus). In this case, immobilization of antibodies had to take place in a different and complementary way than in the case of conventional immunosensors. In this study, three different kinds of immunomolecular-magnetic beads (IMB) were prepared for rapid detection and separation of S. aureus and group G Streptococcus (GGS). The Fc regions of the immobilized antibodies were fully accessible to adsorb protein A or protein G. On the contrary, conventional immunosensors had to have fully accessible Fab regions to facilitate the antigen–antibody recognition. It was suggested that the worse method of immobilization of the antibodies for conventional use would yield the better results for this specific use. In this study, we also perfectly solved the nonspecific adsorptions and interaction problems, which were the most serious critical problems for all kinds of sensors. It was achieved by blocking the excess surface groups of aldehyde IMB and the Fab region of the immobilized antibodies with aldehyde-dextran.     

Spectroscopic characterization of streptavidin functionalized quantum dots

The spectroscopic properties of quantum dots can be strongly influenced by the conditions of their synthesis. In this work, we have characterized several spectroscopic properties of commercial, streptavidin functionalized quantum dots (QD525, lot 1005-0045, and QD585, lot 0905-0031, from Invitrogen). This is the first step in the development of calibration beads to be used in a generalizable quantification scheme of multiple fluorescent tags in flow cytometry or microscopy applications. We used light absorption, photoexcitation, and emission spectra, together with excited state lifetime measurements, to characterize their spectroscopic behavior, concentrating on the 400- to 500-nm wavelength ranges that are important in biological applications. Our data show an anomalous dependence of emission spectrum, lifetimes, and quantum yield (QY) on excitation wavelength that is particularly pronounced in the QD525. For QD525, QY values ranged from 0.2 at 480 nm excitation up to 0.4 at 450 nm and down again to 0.15 at 350 nm. For QD585, QY values were constant at 0.2 between 500 and 400 nm, but they dropped to 0.1 at 350 nm. We attribute the wavelength dependencies to heterogeneity in size and surface defects in the QD525, consistent with characteristics described previously in the chemistry literature. The results are discussed in the context of bridging the gap between what is currently known in the physical chemistry literature of quantum dots and the quantitative needs of assay development in biological applications.     

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.     

Stomach-specific drug delivery of 5-fluorouracil using floating alginate beads

A multiple-unit-type oral floating dosage form (FDF) of 5-fluorouracil (5-FU) was developed to prolong gastric residence time, target stomach cancer, and increase drug bioavailability. The floating bead formulations were prepared by dispersing 5-FU together with calcium carbonate into a mixture of sodium alginate and hydroxypropyl methylcellulose solution and then dripping the dispersion into an acidified solution of calcium chloride. Calcium alginate beads were formed, as alginate undergoes ionotropic gelation by calcium ions and carbon dioxide develops from the reaction of carbonate salts with acid. The evolving gas permeated through the alginate matrix, leaving gas bubbles or pores, which provided the beads buoyancy. The prepared beads were evaluated for percent drug loading, drug entrapment efficiency, image, surface topography, buoyancy, and in vitro release. The formulations were optimized for different weight ratios of gas-forming agent and sodium alginate. The beads containing higher amounts of calcium carbonate demonstrated instantaneous, complete, and excellent floating ability over a period of 24 hours. The optimized formulation was subjected to in vivo antitumor studies to check the therapeutic efficacy of the floating dosage forms containing 5-FU against benzo(a)pyrene-induced stomach tumors in albino female mice (Balb/C strain). The multiple-bead FDF was found to reduce the tumor incidence in mice by 74%, while the conventional tablet dosage form reduced this incidence by only 25%. Results indicate that FDF performed significantly better than the simple tablet dosage form. Published: June 22, 2007     

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.     

A novel binding assay identifies high affinity ligands to the rosiglitazone binding site of mitoNEET

A novel outer mitochondrial membrane protein containing [2Fe-2S] clusters, mitoNEET was first identified through its binding to the anti-diabetic drug pioglitazone. Pioglitazone belongs to a family of drugs that are peroxisome proliferator-activated receptor (PPAR) gamma agonists, collectively known as glitazones. With the lack of pharmacological tools available to fully elucidate mitoNEET's function, we developed a binding assay to probe the glitazone binding site with the aim of developing selective and high affinity compounds. We used multiple thiazolidine-2,4-dione (TZD), 2-thioxothiazolidin-4-one (TTD), and 2-iminothiazolidin-4-one (ITD) compounds to establish several trends to enhance ligand development for the purpose of elucidating mitoNEET function.     

Isolation and characterization of fluorescence-enhancing RNA tags

Methods for the visualization of RNAs are urgently needed for studying a wide variety of cellular processes. Here we report on-bead screening of RNA libraries and its application to the isolation of specific fluorescence-enhancing RNA sequences. A one-bead-one-compound combinatorial RNA library with over one million different sequences was synthesized using the split-and-mix method. Solid-phase synthesis of 30 mer RNAs was performed on 15 ??m and 60 ??m diameter polystyrene beads bearing a non-cleavable linker. The RNA-derivatized beads were incubated with the well-established FlAsH pre-fluorophore and then screened for fluorescence enhancement, either by manually picking the brightest beads under a fluorescence microscope or by sorting with a FACS instrument. A protocol was established for sequence determination from single beads. While numerous RNA sequences showed increased fluorescence when immobilized, only few of them influenced the fluorescence properties of the FlAsH dye when detached from the beads. One of these sequences was found to induce a bathochromic shift in the excitation (from 492 to 510 nm) and emission (from 512 to 523 nm) maxima. This shift was accompanied by a 3.6-fold fluorescence enhancement of FlAsH fluorescence intensity. Mutation studies on the sequence revealed a rather robust structural motif.     

A new genus and species of deep-sea glass sponge (Porifera, Hexactinellida, Aulocalycidae) from the Indian Ocean

New hexactinellid sponges were collected from 2589 m depth on the Carlsberg Ridge in the Indian Ocean during deep-sea dredging. All fragments belong to a new genus and species, Indiellagen. n.ridgenensissp. n., a representative of the family Aulocalycidae described here. The peculiar features of this sponge, not described earlier for other Aulocalycidae, are: longitudinal strands present in several layers and epirhyses channelization.     

Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113

Aims: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. Methods and Results: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113‐based biosensor strains were encapsulated, and their ability to detect 3‐chlorobenzoate (3‐CBA) and 3‐chlorobiphenyl (3‐CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro‐organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3‐CBA or 3‐CBP degradation. In fact, detection of 3‐CBP degradation was enhanced in encapsulated biosensors. Conclusions: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. Significance and Impact of the Study: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.     

Recovery of E,E‐farnesol from cultures of yeast erg9 mutants: Extraction with polymeric beads and purification by normal‐phase chromatography

Saccharomyces cerevisiae erg9 mutants blocked at squalene synthase require ergosterol for growth and produce E,E‐farnesol. Typically, at least half the amount of farnesol remains cell associated. Practically insoluble in water, farnesol can be extracted from production cultures of the erg9 mutants using either methanol/hexane or poly(styrene‐co‐divinylbenzene) beads. The first method consumes more solvents and requires centrifugation to clear an interface emulsion. The second method uses 50% less solvent and the beads can be used repeatedly for extraction. The solvent‐free crude extract from the beads extraction contained higher concentration of farnesol (76–77%) than that from the solvent extraction (61–65%). Farnesol was obtained after normal‐phase chromatography in high overall recovery (94%) and purity (99%). © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009