The increasing use of nanomaterials in healthcare and industrial products heightens the possibility of their ingestion by humans, other mammals, and fish. While toxicity of many nanomaterials has recently been studied, reports of non-lethal effects of nanomaterials remain ill-defined. This study investigates possible pathways by which nanoparticles, titanium dioxide (TiO(2)), could cross the epithelium layer by employing both toxicity and mechanistic studies. This study provides evidence that at 10 microg/mL and above, TiO(2) nanoparticles cross the epithelial lining of the intestinal model by transcytosis, albeit at low levels. TiO(2) was able to penetrate into and through the cells without disrupting junctional complexes, as measured by gamma-catenin. To monitor the epithelial integrity, transepithelial electrical resistance (TEER) was employed and determined low concentrations (10 or 100 microg/mL) of TiO(2) do not disrupt epithelial integrity. Live/dead analysis results did not show cell death after exposure to TiO(2). In addition, at 10 microg/mL (and above) TiO(2) nanoparticles begin alteration of both microvillar organization on the apical surface of the epithelium as well as induce a rise in intracellular-free calcium. The latter is a mechanism cells use to respond to extracellular stimuli and may be linked to the alteration of the apical microvilli. Although TiO(2) does not show cell death, the implication of other, non-lethal, effects could lead to undesired outcomes (i.e., disease, malnutrition, shortened life span, etc.).

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S . Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella -induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

Several isotypes of protein kinase C (PKC) have been reported to be expressed in mammalian eggs, but it is unknown whether these isotypes have a common function in the egg during or within the first few hours of fertilization. Here we show that the isotypes of PKC exhibit distinct patterns of enrichment immediately after mouse egg activation. PKCalpha and gamma accumulate in the egg cortex 25 min post-activation, while only PKCalpha accumulates at the contractile ring of the forming second polar body about 1.5 h post-activation. PKCzeta exhibits some unique features that resulted in it being the focus of more extensive analysis. PKCzeta is tightly associated with the meiotic spindle as determined by detergent extraction and is closely associated with alpha-tubulin as determined by FRET analysis in the metaphase II (MII) egg. In addition, after egg activation, PKCzeta remains associated with the spindle as it transits into anaphase II and later telophase II, becoming associated with the midzone microtubules. Antibodies to the active form of PKCzeta are enriched on the spindle poles and later in development on the midzone microtubules. Active PKCzeta also is enriched in both pronuclei in the 6-h post-fertilization and in the 14-h post-fertilization embryo as well as in the nuclei of the two-cell embryo. Inhibition of PKCzeta, but not inhibition of other isotypes of PKC, results in rapid disruption of the meiotic spindle. This study suggests that PKCzeta has a role in spindle stability, while other PKC isotypes have different roles in the conversion of the egg to the zygote.

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11-member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition.

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