Development and in vitro Evaluation of Antigen‐Loaded Poly(amidoamine) Nanoparticles for Respiratory Epithelium Applications

A poly(amidoamine) with disulfide linkages in the main chain and 4‐hydroxybutyl and ω‐carboxy‐PEG groups (9:1 ratio) as side chains was prepared by Michael addition polymerization of cystamine bisacrylamide with 4‐hydroxybutylamine and ω‐carboxy‐PEG‐amine. To develop therapeutic protein formulations for improved delivery of antigen via the intranasal route, nanoparticles were prepared from this polymer by self‐assembly with p24 or ovalbumin as the model proteins and CpG as the adjuvant. The nanoparticles incorporated the antigens and adjuvant from the feed solution with high efficiency (～90 %) and have sizes of 112 and 169 nm, respectively, with low positive surface charge (～+2 mV). Formulations of the nanoparticles were shown to be nontoxic and stable for at least 10 days at room temperature. Their capacity to pass through epithelial and endothelial cell layers was evaluated in vitro by using a respiratory mucosa‐like barrier model in which monolayers of NCI H441 respiratory epithelial cells and ISO‐HAS‐1 endothelial cells were co‐cultured on both sides of a transwell filter membrane. It was shown that p24 incorporated in the nanoparticles was transported with >140 % greater efficiency through the two contact‐inhibited layers than p24 in its free form, whereas incorporation of ovalbumin in the nanoparticles leads to a 40 % decrease in transport efficiency relative to the free antigen.     

The effect of mercerisation on the electrokinetic potential of cotton

Mercerisation changes the fine structure, morphology and conformation of cotton cellulose chains (cellulose I to cellulose II), resulting in a variation in fibre strength and lustre as well as adsorption properties. Recently it has been shown that mercerisation also changes the electrokinetic behaviour of cotton. The aim of the work presented here is to study the behaviour in unmercerised and mercerised cotton. The zeta potential of cotton fibres was measured by the streaming current method, using an EKA electrokinetic analyser. The relationships between zeta potential and the pH of a potassium chloride solution on the one hand and the point of zero charge (PZC) on the other, determined by titration with a cationic surfactant (cetylpyridinium chloride), were investigated.     

Statistical analysis of intramembranous particles using freeze fracture specimens

We studied the point processes of intramembranous particles of mitochondrial membranes from HeLa cells using the freeze fracture technique. Three groups – under normal conditions, after exposition with rotenone, and after exposition with sodium acid – were compared. First, we used several summary statistics in order to study the two‐dimensional point patterns of intramembranous particles within each group. Then, we compared the patterns in different groups by bootstrap tests using the K‐function and the nearest neighbour distance function G(r). Estimation of the G‐function provided significant results but no significant differences between groups were found using the classical K‐function; estimation of G(r) should therefore not be omitted when studying observed planar point patterns.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased k_cat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208.     

Reverse osmosis desalination plants — marine environmentalist regulator point of view

The environmental characteristics of the brine and its impacts on the marine environment were always the “backyard” and the less concern while planning and operating RO desalination plants. The lack of drinking water in Israel made desalination a national goal. The first huge RO plant was initially operated in Israel on 2005 — 100 Mm³/y and by the end of 2010 305 Mm³/y potable water expected to be produced. Construction and installation of desalination plants requires applying suitable environmental solutions for protecting and preserving the marine and coastal environment from ruin or deterioration.

The Israeli environmental legislation is described including the marine environmental policy and regulations as well as the acquired operational experience during the last two years. Results from the first year of operation and monitoring are shown along with the new appearance of the red phenomenon discharge of VID desalination plant in Ashkelon.

The environmental policy based on the precautionary principle is performed and includes environmental requirements and guidelines for pretreatment, discharge composition, planning marine outfall, background and compliance marine monitoring program and discharge quality standards.     

Self-Assembly Nanoparticles of Natural Bioactive Abietane Diterpenes

Different approaches have been reported to enhance penetration of small drugs through physiological barriers; among them is the self-assembly drug conjugates preparation that shows to be a promising approach to improve activity and penetration, as well as to reduce side effects. In recent years, the use of drug-conjugates, usually obtained by covalent coupling of a drug with biocompatible lipid moieties to form nanoparticles, has gained considerable attention. Natural products isolated from plants have been a successful source of potential drug leads with unique structural diversity. In the present work three molecules derived from natural products were employed as lead molecules for the synthesis of self-assembled nanoparticles. The first molecule is the cytotoxic royleanone 7α-acetoxy-6β-hydroxyroyleanone (Roy, 1) that has been isolated from hairy coleus (Plectranthus hadiensis (Forssk.) Schweinf). ex Sprenger leaves in a large amount. This royleanone, its hemisynthetic derivative 7α-acetoxy-6β-hydroxy-12-benzoyloxyroyleanone (12BzRoy, 2) and 6,7-dehydroroyleanone (DHR, 3), isolated from the essential oil of thicket coleus (P. madagascariensis (Pers.) Benth.) were employed in this study. The royleanones were conjugated with squalene (sq), oleic acid (OA), and/or 1-bromododecane (BD) self-assembly inducers. Roy-OA, DHR-sq, and 12BzRoy-sq conjugates were successfully synthesized and characterized. The cytotoxic effect of DHR-sq was previously assessed on three human cell lines: NCI-H460 (IC₅₀ 74.0 ± 2.2 µM), NCI-H460/R (IC₅₀ 147.3 ± 3.7 µM), and MRC-5 (IC₅₀ 127.3 ± 7.3 µM), and in this work Roy-OA NPs was assayed against Vero-E6 cells at different concentrations (0.05, 0.1, and 0.2 mg/mL). The cytotoxicity of DHR-sq NPs was lower when compared with DHR alone in these cell lines: NCI-H460 (IC₅₀ 10.3 ± 0.5 µM), NCI-H460/R (IC₅₀ 10.6 ± 0.4 µM), and MRC-5 (IC₅₀16.9 ± 0.5 µM). The same results were observed with Roy-OA NPs against Vero-E6 cells as was found to be less cytotoxic than Roy alone in all the concentrations tested. From the obtained DLS results, 12BzRoy-sq assemblies were not in the nano range, although Roy-OA NP assemblies show a promising size (509.33 nm), Pdl (0.249), zeta potential (−46.2 mV), and spherical morphology from SEM. In addition, these NPs had a low release of Roy at physiological pH 7.4 after 24 h. These results suggest the nano assemblies can act as prodrugs for the release of cytotoxic lead molecules.