Eye drop delivery of nano-polymeric micelle formulated genes with cornea-specific promoters

BACKGROUND: This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms. METHODS: Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide. RESULTS: The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment. CONCLUSIONS: Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport.     

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.     

Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process

A shortcut biological nitrogen removal (SBNR) utilizes the concept of a direct conversion of ammonium to nitrite and then to nitrogen gas. A successful SBNR requires accumulation of nitrite in the system and inhibition of the activity of nitrite oxidizers. A high concentration of free ammonia (FA) inhibits nitrite oxidizers, but unfortunately decreases the ammonium removal rate as well. Therefore, the optimal range of FA concentration is necessary not only to stabilize nitrite accumulation but also to achieve maximum ammonium removal. In order to derive such optimal FA concentrations, the specific substrate utilization rates of ammonium and nitrite oxidizers were measured. The optimal FA concentration range appeared to be 5–10 mg/L for the adapted sludge. The simulated results from the modified inhibition model expressed by FA and ammonium/nitrite concentrations were shown very similar to the experimental results.     

THE UTILITY OF PROTEOMICS IN ALGAL TAXONOMY: BOSTRYCHIA RADICANS/B. MORITZIANA (RHODOMELACEAE,RHODOPHYTA) AS A MODEL STUDY1

A comparison of the proteome of eight genetically well‐characterized isolates of the Bostrychia radicans (Mont.) Mont./B. moritziana (Sond. ex Kütz.) J. Agardh species complex was undertaken to establish if genetic relationships among them can be determined using proteome data. Genetic distances were calculated on the basis of common and distinct spots in two‐dimensional gel electrophoresis (2‐DE). Proteomes of the male and female plants of each population were compared to analyze the range of genetic difference within an isolate. Haploid male and female plants of the same species had 3.7%–7.1% sex‐specific proteins. The degree of similarity of the proteome was consistent with previous DNA sequence data and sexual compatibility studies between the isolates. Two sexually compatible isolates from Venezuela showed a pair‐wise distance ranging from 0.14 to 0.21. The isolates from Mexico and Venezuela, which were partially compatible, showed a maximum pair‐wise distance of 0.26. A high level of genetic difference was found among isolates that were sexually incompatible. The isolate from Brazil was reproductively isolated from the Mexico and Venezuela isolates and showed a maximum pair‐wise distance of 0.65 and 0.58, respectively. Comparative proteomics may be helpful for studying genetic distances among algal samples, if intraisolate variation (gene expression) can be minimized or tested.     

The adhesion of thin carbon films to metallic substrates.

As part of the development of carbon-coated prosthetic devices, the adhesion of thin carbon films to metallic substrates has been studied. The bond strength of carbon films about 5000 A thick on Ti-6A1-4V and stainless steel was measured in a pull test and found to be greater than 4700 psi. Auger electron spectroscopy showed a reactive film/substrate interface. The ultimate bond strength was found to be dependent on the substrate and the deposition parameters.     

A phylogenetic analysis of rissooidean and cingulopsoidean families (Gastropoda: Caenogastropoda)

The Rissooidea is one of the largest and most diverse molluscan superfamilies, with 23 recognized Recent families including marine, freshwater and terrestrial members. The Cingulopsoidea are a group of three marine families previously included within the Rissooidea. A previous molecular analysis including two rissooideans and one cingulopsoidean, indicated the possibility that the Rissooidea is at least diphyletic. We use new molecular data to investigate the polyphyly of Rissooidea and test the monophyly of Cingulopsoidea with a greatly increased taxon set. This study includes the greatest sampling to date with 43 species of 14 families of Rissooidea and all families of Cingulopsoidea. Bayesian and maximum likelihood analyses of 16S and 28S show that there are two major clades encompassing taxa previously included in Rissooidea. These are the Rissooidea s.s. containing Rissoidae and Barleeiidae and the Truncatelloidea containing Anabathridae, Assimineidae, Falsicingulidae, Truncatellidae, Pomatiopsidae, Hydrobiidae s.l., Hydrococcidae, Stenothyridae, Calopiidae, Clenchiellidae, Caecidae, Tornidae, and Iravadiidae. Rissoidae is not monophyletic, with Lironoba grouping with Emblanda (Emblandidae) and Rissoina forming a separate clade with Barleeiidae. Iravadiidae is not monophyletic, with Nozeba being sister to the Tornidae. Tatea, usually included within Hydrobiidae, is distinct from that family and Nodulus, previously included in Anabathridae, groups with the hydrobiids.