Multicenter Clinical Validation of a Cartridge-Based Real-Time PCR System for Detection of Coccidioides spp. in Lower Respiratory Specimens.

Available methods for the diagnosis of coccidioidomycosis have significant shortcomings relative to accuracy and timeliness. We retrospectively and prospectively evaluated the diagnostic performance and reproducibility of a new cartridge-based real-time PCR assay for Coccidioides spp. directly in lower respiratory secretions and compared them to today's "gold standard," fungal culture. The GeneSTAT Coccidioides assay uses a 106-bp target sequence repeated multiple times (∼60×) per genome, thus lowering the limit of detection (LOD) for extracted DNA to 10 genome equivalents/ml. A total of 332 prospective and retrospective individual patient specimens were tested. The retrospective samples consisted of 100 bronchoalveolar lavage or bronchial wash (BAL/BW) (51 positive and 49 negative by culture) specimens that had been collected previously and stored at -70°C. These samples were tested by the GeneSTAT Coccidioides assay across three clinical test sites. The sensitivity was 100%, and the specificity ranged between 93.8% and 100%. There was minimal variance in the percent agreement across the three sites, 95.6% to 100%. Additionally, a total of 232 fresh (prospective) deidentified BAL/BW specimens were tested across the three clinical sites, which included a number of specimens from Southern California to provide a diversity of isolates. Specimens were tested by fungal culture, with any isolates of Coccidioides, except for one, being confirmed by molecular means (AccuProbe). The sensitivity of the GeneSTAT Coccidioides assay across the three sites was 100% (4/4) for positive fresh specimens, and the overall specificity of the assay was 99.6% (227/228), ranging from 98.1% to 100%. In testing for cross-reactivity, the assay was 100% specific when screened against 47 different bacterial, viral, and fungal species.

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform.

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.

Commenting on the effects of surface treated- and non-surface treated TiO(2) in the Caco-2 cell model.

In a recent work published in Particle and Fibre Toxicology by Fisichella and coworkers investigating surface-modified TiO2 nanoparticle exposure in a model human intestinal epithelium (Caco-2), albeit degraded to mimic conditions in the gut and exposure to natural sunlight, purportedly resulted in no toxic effects. The authors (Fisichella et al.) claim to have confirmed the results of a 2010 report by Koeneman et al. However, the study by Koeneman and colleagues revealed significant effects of unmodified TiO2 nanoparticles. These contradicting data warrant further investigation into the possible effects of aluminum hydroxide, as these nanoparticles appear to have resulted in an abnormal apical surface in Caco-2 cells.

Toxicity and cellular responses of intestinal cells exposed to titanium dioxide.

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.).

Involvement of the PKC family in regulation of early development.

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.

Experimental approach for an in vitro toxicity assay with non-aggregated quantum dots.

Engineered nanoparticles are increasingly used in consumer products. While the potential of these products hold great promise, it is not known what potential toxic effects these nanomaterials may have on human health. There is a need to develop affordable, systematic, short-term in vitro assays aimed at allowing rapid assessment of potential toxicity. The method reported in this paper describes a system in which the intestinal lining is mimicked (Caco-2 human intestinal cell line) and provides an environment in which quantum dots (QDs), and possibly other nanomaterials, can be applied. Transepithelial electrical resistance (TEER) measurements assessed whether the epithelial integrity was breached because of QD exposure. QDs were suspended in calcium/magnesium-free phosphate buffered saline to study non-aggregated QDs. To maintain cell integrity, normal cell culture conditions were retained below the epithelium to provide necessary nutrients and ions. Toxicity studies completed here show that the nanosized QDs coated with hydrophilic thioglycolate capping ligands purchased for these experiments caused disruption in the epithelium monolayer and cell death at 0.1mg/L of QDs. This toxicity was caused by the nano-size of the QDs rather than the cadmium ions or the sodium thioglycolate capping ligands. Aggregated QDs did not cause toxicity as measured by TEER.

PKC isotypes in post-activated and fertilized mouse eggs: association with the meiotic spindle.

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.

An ex vivo method for evaluating the biocompatibility of neural electrodes in rat brain slice cultures.

Failure of neural recording electrodes implanted in the brain is often attributed to the formation of glial scars around the implant. A leading cause of scar formation is the electrode material. Described below is an approach to evaluate the biocompatibility of novel electrode materials in a representative three-dimensional model. The model, brain slice culture, accounts for the response of the neural tissue in the absence of the systemic response. While limitations of any in vitro model exist, brain slice culture provides an indication of the response of neurons and glia in an environment more indicative of the in vivo environment than two-dimensional cell culture of glia or neurons alone. Polybenzylcyclobutene (BCB) electrodes were developed as test materials for flexible electrodes due to ease of processing, low water uptake, and inherent flexibility when formed in thin sheets. Biocompatibilty of the BCB neural electrodes was evaluated using living brain slices derived from the hippocampal regions of 100 g CD rats. Importantly, fewer animals can be used in brain slice culture to evaluate the neural tissue response than when using live animals, since several slices can be obtained per animal. Cellular response to the electrodes was evaluated at 0, 7, and 14 days. At all time points living cells, both neurons and glia, were observed in the vicinity of the electrode. In addition, cells were observed migrating out from the brain slices onto the shank of the BCB electrode. Brain slice culture is shown to be a viable alternative to in vivo evaluation, in that the response of both neurons and glia can be evaluated in a native three-dimensional state, while sacrificing fewer animals. Future in vivo evaluation with BCB will provide definitive answers on the degree of glial scarring in response to this new and biocompatible electrode material.