Amplification of complex gene libraries by emulsion PCR.
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Emulsions↗
Biomedical subjects
Shlomo Magdassi
Publications and source records attributed to Shlomo Magdassi.
Directed evolution by in vitro compartmentalization.
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Cell Compartmentation↗
Ring stain effect at room temperature in silver nanoparticles yields high electrical conductivity.
We demonstrate that metallic rings formed spontaneously at room temperature via evaporation of aqueous drops containing silver nanoparticles (20-30 nm in diameter) exhibit high electrical conductivity (up to 15% of that for bulk silver). The mechanism underlying this self-assembly phenomena is the "ring stain effect", where self-pinning is combined with capillary flow to form a ring consisting of close-packed metallic nanoparticles along the perimeter of a drying droplet. Our macroscopic and microscopic (applying conductive atomic force microscopy) transport measurements show that the conductivity of the ring, which has a metallic brightness, is orders of magnitude larger than that of corresponding aggregates developed without the ring formation, where high conductivity is known to appear only after annealing at high temperature.
Letter↗
Solubilization of hydrophobic molecules in nanoparticles formed by polymer-surfactant interactions.
The interaction between the anionic surfactant, sodium dodecyl sulfate, and the polyelectrolyte, poly(diallyldimethylammonium chloride), may lead to formation of nanoparticles dispersed in water. The morphology of the resulting nanoparticles and their ability to solubilize hydrophobic molecules were evaluated. As shown by SEM and AFM imaging, the particles are spherical, having a diameter of about 20 nm. The solubilization within the nanoparticles was tested with pyrene, a fluorescence probe, and Nile Red, a solvatochromic probe. It was found that for Nile Red the solubilization within the nanoparticles is at lower polarity than for SDS micelles, and from pyrene solubilization it appears that the hydrophobicity of the nanoparticles depends on the ratio between the SDS molecules and the charge unit of the polymer.
Hydrophobic and Hydrophilic Interactions↗
High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments.
Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >10(4) enzyme molecules, in a volume of <10 femtoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.
Aryldialkylphosphatase↗
Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery.
In vitro compartmentalization (IVC) uses water-in-oil emulsions to create artificial cell-like compartments in which genes can be individually transcribed and translated. Here, we present a new application of IVC for the selection of DNA-nuclease inhibitors. We developed a nano-droplets delivery system that allows the transport of various solutes, including metal ions, into the emulsion droplets. This transport mechanism was used to regulate the activity of colicin nucleases that were co-compartmentalized with the genes, so that the nucleases were activated by nickel or cobalt ions only after the potential inhibitor genes have been translated. Thus, genes encoding nuclease inhibitors survived the digestion and were subsequently amplified and isolated. Selection is therefore directly for inhibition, and not for binding of the nuclease. The stringency of selection can be easily modulated to give high enrichments (100-500-fold) and recoveries. We demonstrated its utility by selecting libraries of the gene encoding the cognate inhibitor of colicin E9 (immunity protein 9, or Im9) for inhibition of another colicin (ColE7). The in vitro evolved inhibitors show significant inhibition of ColE7 both in vitro and in vivo. These Im9 variants carry mutations into residues that determine the selectivity of the natural counterpart (Im7) while completely retaining the residues that are conserved throughout the family of immunity protein inhibitors. The in vitro evolution process confirms earlier hypotheses regarding the "dual recognition" binding mechanism and the way in which new colicin-immunity pairs diverged from existing ones.
Colicins↗
Investigation on the release of fluorescent markers from w/o/w emulsions by fluorescence-activated cell sorter.
The mechanism of release of two fluorescent markers, fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) and fluorescein, from water-in-oil-in-water (w/o/w) emulsions was investigated using a rapid and sensitive method based on fluorescence-activated cell sorting (FACS). The release of FITC-BSA from a w/o/w emulsion was controlled by diffusion rather than by simple breakdown of the multiple droplets or by formation of reverse micelles in the oil phase. In contrast, the release of fluorescein from a double emulsion was controlled by formation of reverse micelles rather than by diffusion or simple breakdown of multiple droplets. A significant difference in the yield and fraction of FITC-BSA and fluorescein released from double emulsions was observed due to their different molecular structure and properties. The yield of FITC-BSA incorporation in a double emulsion increased with increasing FITC-BSA concentration in the internal water phase, while the yield of fluorescein decreased with increasing concentration. The fraction of FITC-BSA released from a w/o/w emulsion after 24 h decreased with an increasing concentration of FITC-BSA in the internal phase. The w/o/w emulsion with internalized FITC-BSA was more stable than that with fluorescein, indicating its further application for sorting or enriching size-controlled double droplets that contained genes and water-soluble drugs.
Algorithms↗
Liposome transduction into cells enhanced by haptotactic peptides (Haptides) homologous to fibrinogen C-termini.
Haptides are 19-21mer cell-binding peptides equivalent to sequences on the C-termini of fibrinogen beta chain (Cbeta), gamma chain (preCgamma) and the extended alphaE chain of fibrinogen (CalphaE). In solution, Haptides accumulated in cells by non-saturable kinetics [Exp. Cell Res. 287 (2003) 116]. This study describes Haptide interactions with liposomes and Haptide-mediated liposome uptake by cells. Haptides became incorporated into negatively charged liposomes, changing their zeta potential. Atomic force microscopy and particle sizing by light scattering showed that the liposomes dissolved Haptide nanoparticles and absorbed them from solution. Pre-mixing fluorescent rhodamine-containing liposomes or "stealth" doxorubicin (DOX)-containing liposomes (Doxil) with Cbeta, preCgamma or to a lesser degree CalphaE, significantly enhanced their uptake by fibroblasts and endothelial cells. Confocal microscopy showed Haptide-induced liposome uptake saturated above approximately 40 microM Haptide. Cytotoxicity tests with lower concentrations of Doxil liposomes indicated that premixing with approximately 40 microM Cbeta or preCgamma increased their toxicity by one order of magnitude. It was evident that the liposomes complexed with an amphiphilic Haptide are transduced through cell membranes, probably by a non-receptor-mediated process. These results suggest that Cbeta or pre-Cgamma could be employed to augment the cellular uptake of drugs in liposomal formulations.
Animals↗
Flow cytometry: a new method to investigate the properties of water-in-oil-in-water emulsions.
We present a new and facile method to evaluate w/o/w emulsions containing fluorescent markers by flow cytometry. Flow cytometry allows simultaneous measurement of w/o/w emulsion droplets "marked" with a fluorescent marker or "blank" without the need for complicated sample preparation. The yield of preparation of the w/o/w emulsion and the release rate of the fluorescent marker FITC-BSA were investigated by this new method. The release fraction (after 24 h) of FITC-BSA from the w/o/w emulsion decreased with increasing concentration of FITC-BSA inside the internal phase, just like the release fraction of NaCl as marker from the w/o/w emulsion. Flow cytometry results show that the yield and release behavior in w/o/w emulsions are in agreement with results reported by more complicated methods.
Journal Article↗
In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.
Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes.
DNA Modification Methylases↗
Repellency of citronella for head lice: double-blind randomized trial of efficacy and safety.
BACKGROUND: Head lice move easily from head to head. The lack of safe, effective repellents leads to reinfestation. OBJECTIVES: To test the efficacy of a slow-release citronella formulation as a repellent against the head louse. METHODS: During 4 months in 2003 a randomized, placebo-controlled double-blind clinical study was conducted in four elementary schools; 103 children were treated with the test formulation and 95 with a placebo. RESULTS: A significant difference was observed during the second examination 2 months later, when 12.0% of the children treated with the test repellent and 50.5% of those treated with placebo were infested with lice. A significant difference was also observed at the third examination 2 months later, when 12.4% of the children treated with the test repellent and 33.7% treated with placebo were infested. Overall, there were significant differences between those treated with the repellent and those treated with the placebo (15.4% and 55.1% respectively, P < 0.0001). Side effects were observed in 4.4% of children who disliked the odor of the formulation, and an additional 1.0% who complained of a slight itching and burning sensation. CONCLUSIONS: Use of an effective repellent could significantly lower the incidence of reinfestations, which would lower expenditure on lice control, including pediculicides, combs and products for nit removal, and the time spent on treatment and removal of the nits.
Adolescent↗
Photostabilization of an entomopathogenic fungus using composite clay matrices.
To provide photostabilization for entomopathogenic fungi by anionic dyes, composite matrices based on clay-biopolymer combinations were prepared. In the first step, the negative surface charge of various clays (montmorillonite, attapulgite, bentonite and kaolinite) was reversed to positive by adsorption to the polycationic biopolymer chitosan. The second step involved adsorption of the toxicologically safe anionic dyes fast green (FG) and naphthol yellow S (NYS) to the clay complexes. Compared with cytotoxic photoprotectants like berberine, palmatine and acriflavine, the anionic dyes have no adverse effects up to a concentration of 1 M. In assays using various clay-chitosan-dye matrices and UV irradiation from a lamp source, it was evident that both FG and NYS provided considerable photostabilization for conidia of the entomopathogenic fungus Aschersonia spp. that served as a model biocontrol agent. Apparently, because of the light-dispersing property, bentonite and attapulgite per se provided significant photoprotection. All clay matrices containing FG provided a substantial photostabilization effect.
Aluminum Silicates↗
Water-soaked evidence: detectability of explosive traces after immersion in water.
Various factors governing the detectability of explosive traces after being soaked in water were studied. The variables are: the type of the surface (surfaces liable to be found in aircraft were chosen), the type of explosive, the type of water (tap or seawater), and movement of the immersed surface in the water. The maximal immersion times (tmax) after which explosive detection was possible were evaluated. This datum was found to depend on the type of explosive (one of the important factors is solubility in water), the surface material and the environmental conditions (tap or seawater movement). Detection of PETN on high-density polyethylene, linoleum, glass and aluminum, by the chemical Explosive Testing Kit (ETK), was possible even after a month of soaking in seawater. In addition, it has been found that movement of bulk water around the samples with deposited explosives considerably decreases tmax values. It is, therefore, recommended to retrieve samples for explosive analysis as soon as possible and in areas where the currents of water is minimal.
Journal Article↗