Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles. (C) 2004 Elsevier Inc. All rights reserved.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

New magnetic nano-absorbent for the determination of n-octanol/water partition coefficients

A novel and generic miniaturization methodology for the determination of partition coefficient values of organic compounds in noctanol/water by using magnetic nanoparticles is, for the first time, described. We have successfully designed, synthesised and characterised new colloidal stable porous silica-encapsulated magnetic nanoparticles of controlled dimensions. These nanoparticles absorbing a tiny amount of n-octanol in their porous silica over-layer are homogeneously dispersed into a bulk aqueous phase (pH 7.40) containing an organic compound prior to magnetic separation. The small size of the particles and the efficient mixing allow a rapid establishment of the partition equilibrium of the organic compound between the solid supported n-octanol nano-droplets and the bulk aqueous phase. UV-vis spectrophotometry is then applied as a quantitative method to determine the concentration of the organic compound in the aqueous phase both before and after partitioning (after magnetic separation). log D values of organic compounds of pharmaceutical interest (0.65-3.50), determined by this novel methodology, were found to be in excellent agreement with the values measured by the shake-flask method in two independent laboratories, which are also consistent with the literature data. It was also found that this new technique gives a number of advantages such as providing an accurate measurement of log D value, a much shorter experimental time and a smaller sample size required. With this approach, the formation of a problematic emulsion, commonly encountered in shake-flask experiments, is eliminated. It is envisaged that this method could be applicable to the high throughput log D screening of drug candidates. (c) 2005 Elsevier B.V. All rights reserved.

A new porous 2D coordination polymer built by copper(II) and trimesic acid

A 2D porous material, Cu-3(tmen)(3)(tma)(2)(H2O)(2)(.)6.5H(2)O [tmen = N,N,N',N'-tetramethylethane-1,2-diamine; tmaH(3) = 1,3,5-benzenetricarboxylic acid/trimesic acid], has been synthesized and characterized by X-ray single crystal analysis, variable temperature magnetic measurements, IR spectra and XRPD pattern. The complex consists of 2D layers built by three crystallographically independent Cu(tmen) moieties bridged by tma anions. Of the three copper ions, Cu(1) and Cu(2) present distorted square pyramidal coordination geometry, while the third exhibits a severely distorted octahedral environment. The Cu(1)(tmen) and Cu(2)(tmen) building blocks bridged by tma anions give rise to chains with a zig-zag motif, which are cross-connected by Cu(3)(tmen)-tma polymers sharing metal ions Cu(2) through pendant tma carboxylates. The resulting 2D architecture extends in the crystallographic ab-plane. The adjacent sheets are embedded through the Cu(3)(tmen) tma chains, leaving H2O-filled channels. There are 6.5 lattice water molecules per formula unit, some of which are disordered. Upon heating, the lattice water molecules get eliminated without destroying the crystal morphology and the compound rehydrated reversibly on exposure to humid atmosphere. Magnetic data of the complex have been fitted considering isolated irregular Cu-3 triangles (three different J parameters) by applying the CLUMAG program. The best fit indicates three close comparable J parameters and very weak antiferromagnetic interactions are operative between the metal centers. (C) 2004 Elsevier B.V. All rights reserved.

Preparation of nanomagnetic absorbent for partition coefficient measurement

In this paper, we report a new method based on supercritical carbon dioxide (scCO(2)) to fill and distribute the porous magnetic nanoparticles with n-octanol in a homogeneous manner. The high solubility of n-octanol in scCO(2) and high diffusivity and permeability of the fluid allow efficient delivery of n-octanol into the porous magnetic nanoparticles. Thus, the n-octanol-loaded magnetic nanoparticles can be readily dispersed into aqueous buffer (pH 7.40) to form a homogenous suspension consisting of nano-sized n-octanol droplets. We refer this suspension as the n-octanol stock solution. The n-octanol stock solution is then mixed with bulk aqueous phase (pH 7.40) containing an organic compound prior to magnetic separation. The small-size of the particles and the efficient mixing enable a rapid establishment of the partition equilibrium of the organic compound between the solid supported n-octanol nano-droplets and the bulk aqueous phase. UV-vis spectrophotometry is then applied to determine the concentration of the organic compound in the aqueous phase both before and after partitioning (after magnetic separation). As a result, log D values of organic compounds of pharmaceutical interest determined by this modified method are found to be in excellent agreement with the literature data. (c) 2006 Elsevier B.V. All rights reserved.

Synthesis of well-dispersed nanoparticles within porous solid structures using surface-tethered surfactants in supercritical CO2

We have developed a new method for the synthesis of Pd nanoparticles with controllable sizes within a silica matrix using solid-supported surfactants in supercritical CO2. XRD, HRTEM and CO chemisorption data show that unformly sized Pd nanoparticles are evenly distributed within the porous silica and are chemically tethered by surfactant molecules [poly(oxyethylene stearyl ether) and fluorinated poly(oxyethylene)]. It is postulated that tiny solid-supported surfactant assemblies act as nano-reactors for the template synthesis of nanoparticles or clusters from the soluble precursors therein.

Aerogel-coated metal nanoparticle colloids as novel entities for the synthesis of defined supported metal catalysts

Nanometer metal particles of tailored size (3-5 nm) and composition prepared via inverse microemulsion were encapsulated by ultrathin coatings (<2.5 nm) of inorganic porous aerogels covered with surface -OH groups. These composite materials formed metastable colloids in solvent(s), and the organic surfactant molecules were subsequently removed without leading to aggregation (the ethanolic colloid solution was shown to be stable against flocculation for at least weeks). We demonstrate that the totally inorganic-based composite colloids, after the removal of surfactant, can be anchored to conventional solid supports (gamma-alumina, carbons) upon mixing. Application of a high temperature resulted in the formation of strong covalent linkages between the colloids and the support because of the condensation of surface groups at the interface. Detailed characterizations (X-ray diffraction (XRD), pore analysis, transmission electron microscopy (TEM), CO chemisorption) and catalytic testing (butane combustion) showed that there was no significant metal aggregation from the fine metal particles individually coated with porous aerogel oxide. Most of these metal sites on the coated nanoparticles with and without support are fully accessible by small molecules hence giving extremely active metal catalysts. Thus, the product and technology described may be suitable to synthesize these precursor entities of defined metal sizes (as inks) for wash coat/impregnation applications in catalysis. The advantages of developing inorganic nanocomposite chemical precursors are also discussed.

Carbon fractions associated with silt-size particles in surface and subsurface soil horizons

Information on the distribution and behavior of C fractions in soil particle sizes is crucial for understanding C dynamics in soil. At present little is known about the behavior of the C associated with silt-size particles. We quantified the concentrations, distribution, and enrichment of total C (TC), readily oxidizable C (ROC), hotwater- extractable C (HWC), and cold-water-extractable C (CWC) fractions in coarse (63–20-mm), medium (20–6.3-mm), and fine (6.3–2-mm) silt-size subfractions and in coarse (2000–250 mm) and fine (250–63 mm) sand and clay (<2-mm) soil fractions isolated from bulk soil (<2 mm), and 2- to 4-mm aggregate-size fraction of surface (0–25 cm) and subsurface (25–55 cm) soils under different land uses. All measured C fractions varied significantly across all soil particle-size fractions. The highest C concentrations were associated with the <20-mm soil fractions and peaked in the medium (20–6.3-mm) and fine (6.3–2-mm) silt subfractions in most treatments. Carbon enrichment ratios (ERC) revealed the dual behavior of the C fractions associated with the medium silt-size fraction, demonstrating the simultaneous enrichment of TC and ROC, and the depletion of HWC and CWC fractions. The medium silt (20–6.3-mm) subfraction was identified in this study as a zone where the associated C fractions exhibit transitory qualities. Our results show that investigating subfractions within the silt-size particle fraction provides better understanding of the behavior of C fractions in this soil fraction.

Assessing the impact of nano- and micro-scale zerovalent iron particles on soil microbial activities: Particle reactivity interferes with assay conditions and interpretation of genuine microbial effects

The effects of nano-scale and micro-scale zerovalent iron (nZVI and mZVI) particles on general (dehydrogenase and hydrolase) and specific (ammonia oxidation potential, AOP) activities mediated by the microbial community in an uncontaminated soil were examined. nZVI (diameter 12.5 nm; 10 mg gÿ1 soil)apparently inhibited AOP and nZVI and mZVI apparently stimulated dehydrogenase activity but had minimal influence on hydrolase activity. Sterile experiments revealed that the apparent inhibition of AOP could not be interpreted as such due to the confounding action of the particles, whereas, the nZVIenhanced dehydrogenase activity could represent the genuine response of a stimulated microbial population or an artifact of ZVI reactivity. Overall, there was no evidence for negative effects of nZVI or mZVI on the processes studied. When examining the impact of redox active particles such as ZVI on microbial oxidation–reduction reactions, potential confounding effects of the test particles on assay conditions should be considered.

Olive oil increases the number of triacylglycerol-rich chylomicron particles compared with other oils: an effect retained when a second standard meal is fed

Background: Compared with the postprandial events after a single meal, different events occur when a second meal is ingested 4–6 h after a first meal. There is a rapid appearance of chylomicrons in the circulation carrying fat ingested with the first meal, with a peak 1 h after the second meal. Objective: Our goal was to examine whether different dietary oils have effects on the storage of triacylglycerol as a result of differences in their digestion, absorption, and incorporation into chylomicrons. Design: A single-blind, randomized, within-subject crossover design was used to study the effects of palm oil, safflower oil, a mixture of fish and safflower oil, and olive oil on postprandial apolipoprotein (apo) B-48, retinyl ester, and triacylglycerol in the Sf > 400 fraction with the use of a sequential meal protocol. Results: For triacylglycerol, retinyl ester, and apo B-48, the time to reach peak concentration was significantly earlier after the second meal than after the first meal (P < 0.005). This was apparent with each of the dietary oils. The pattern of the apo B-48 response differed significantly among the dietary oils, with olive oil resulting in higher concentrations after both meals (P = 0.003). The ratio of triacylglycerol to apo B-48 was significantly lower after olive oil feeding than after feeding with the other oils (P = 0.02). Conclusions: The rapid entry of chylomicrons after the ingestion of a second meal 5 h after a first meal was seen with all of the oils investigated. The short-term ingestion of olive oil produced more chylomicrons than did the other dietary oils, which may have been due to differences in the metabolic handling of olive oil within the gut.

Chemical ageing and transformation of diffusivity in semi-solid multi-component organic aerosol particles

Recent experimental evidence underlines the importance of reduced diffusivity in amorphous semi-solid or glassy atmospheric aerosols. This paper investigates the impact of diffusivity on the ageing of multi-component reactive organic particles representative of atmospheric cooking aerosols. We apply and extend the recently developed KM-SUB model in a study of a 12-component mixture containing oleic and palmitoleic acids. We demonstrate that changes in the diffusivity may explain the evolution of chemical loss rates in ageing semi-solid particles, and we resolve surface and bulk processes under transient reaction conditions considering diffusivities altered by oligomerisation. This new model treatment allows prediction of the ageing of mixed organic multi-component aerosols over atmospherically relevant time scales and conditions. We illustrate the impact of changing diffusivity on the chemical half-life of reactive components in semisolid particles, and we demonstrate how solidification and crust formation at the particle surface can affect the chemical transformation of organic aerosols.

Unit-cell approximation for diblock−copolymer brushes grafted to spherical particles

This paper examines the equilibrium phase behavior of thin diblock-copolymer films tethered to a spherical core, using numerical self-consistent field theory (SCFT). The computational cost of the calculation is greatly reduced by implementing the unit-cell approximation (UCA) routinely used in the study of bulk systems. This provides a tremendous reduction in computational time, permitting us to map out the phase behavior more extensively and allowing us to consider far larger particles. The main consequence of the UCA is that it omits packing frustration, but evidently the effect is minor for large particles. On the other hand, when the particles are small, the UCA calculation can be readily followed up with the full SCFT, the comparison to which conveniently allows one to quantitatively assess the effect of packing frustration.

Using pH abnormalities in diseased skin to trigger and target topical therapy

Abstract Purpose: The pH discrepancy between healthy and atopic dermatitis skin was identified as a site specific trigger for delivering hydrocortisone from microcapsules. Methods: Using Eudragit L100, a pH-responsive polymer which dissolves at pH 6, hydrocortisone-loaded microparticles were produced by oil-in-oil microencapsulation or spray drying. Release and permeation of hydrocortisone from microparticles alone or in gels was assessed and preliminary stability data was determined. Results: Drug release from microparticles was pH-dependent though the particles produced by spray drying also gave significant non-pH dependent burst release, resulting from their porous nature or from drug enrichment on the surface of these particles. This pH-responsive release was maintained upon incorporation of the oil-in-oil microparticles into Carbopol- and HPMC-based gel formulations. In-vitro studies showed 4 to 5-fold higher drug permeation through porcine skin from the gels at pH 7 compared to pH 5. Conclusions: Permeation studies showed that the oil-in-oil generated particles deliver essentially no drug at normal (intact) skin pH (5.0 – 5.5) but that delivery can be triggered and targeted to atopic dermatitis skin where the pH is elevated. The incorporation of these microparticles into Carbopol- and HPMC-based aqueous gel formulations demonstrated good stability and pH-responsive permeation into porcine skin.

Chemical ageing and transformation of diffusivity in semi-solid multi-component organic aerosol particles

Recent experimental evidence underlines the importance of reduced diffusivity in amorphous semi-solid or glassy atmospheric aerosols. This paper investigates the impact of diffusivity on the ageing of multi-component reactive organic particles approximating atmospheric cooking aerosols. We apply and extend the recently developed KMSUB model in a study of a 12-component mixture containing oleic and palmitoleic acids. We demonstrate that changes in the diffusivity may explain the evolution of chemical loss rates in ageing semi-solid particles, and we resolve surface and bulk processes under transient reaction conditions considering diffusivities altered by oligomerisation. This new model treatment allows prediction of the ageing of mixed organic multi-component aerosols over atmospherically relevant timescales and conditions. We illustrate the impact of changing diffusivity on the chemical half-life of reactive components in semi-solid particles, and we demonstrate how solidification and crust formation at the particle surface can affect the chemical transformation of organic aerosols.