A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

Steric barriers such as collagen I sharply limit interstitial delivery of macromolecular and nanoparticle (NP) based therapeutic agents. Collagenase-linked superparamagnetic NPs overcame these barriers and moved through in vitro extracellular matrix (ECM) at 90 microm h(-1), a rate similar to invasive cells, under the influence of a magnetic field. NP migration in ECM diminished linearly over 5 days. The collagenase-NP construct overcame two of the most significant barriers to nano- and microscale therapeutics deployment: proteolytic enzyme stability was maintained during a clinically useful time frame by immobilization on the NP surface and degradation of interstitial barriers to tissue biodistribution was enabled by the conjugated microbial protease.

Controlled dispersion of therapeutic agents within liquid- and gel-filled cavities represents a barrier to treatment of some cancers and other pathological states. Interstitial delivery is compromised by the poor mobility of macromolecules and larger nanoscale structures. We developed an in vitro system to quantify the suitability of superparamagnetic nanoparticles (SPM NPs) as a site-specific therapeutic vehicle for delivery through fluid- and gel-based systems. SPM NP motion was induced by an external magnetic field. NP migration was modulated by NP concentration and surface coating. 135 nanometer radius PEGylated NPs moved through the extracellular matrix with an average velocity of 1.5 mm h(-1), suitable for some clinical applications. Increasing the SPM NP radius to 400 nm while maintaining the same per NP magnetic susceptibility resulted in a greater than 1,000-fold reduction in magnetic mobility, to less than 0.01 mm h(-1). The critical influence of NP size on gel permeation was also observed in silica-coated 135 nm SPM NPs that aggregated under the experimental conditions. Aggregation played a critical role in determining the behavior of the nanoparticles. SPM NPs allow significant free-solution mobility to specific sites within a cavity and generate sufficient force to penetrate common in vivo gels.

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Respiratory syncytial virus (RSV) is a major cause of pneumonia and wheezing in infants and the elderly, but to date there is no licensed vaccine. We developed a gold nanorod construct that displayed the major protective antigen of the virus, the fusion protein (F). Nanorods conjugated to RSV F were formulated as a candidate vaccine preparation by covalent attachment of viral protein using a layer-by-layer approach. In vitro studies using ELISA, confocal microscopy and circular dichroism revealed that conformation-dependent epitopes were maintained during conjugation, and transmission electron microscopy studies showed that a dispersed population of particles could be achieved. Human dendritic cells treated with the vaccine-induced immune responses in primary human T cells. These results suggest that this vaccine approach may be a potent method for immunizing against viruses such as RSV with surface glycoproteins that are targets for the human immune response.

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One of the greatest goals of neuroscience in recent decades has been to rehabilitate individuals who no longer have a functional relationship between their mind and their body. Although neuroscience has produced technologies which allow the brains of paralyzed patients to accomplish tasks such as spell words or control a motorized wheelchair, these technologies utilize parts of the brain which may not be optimal for simultaneous use. For example, if you needed to look at flashing lights to spell words for communication, it would be difficult to simultaneously look at where you are moving. To improve upon this issue, this study developed and tested the foundation for a speech prosthesis paradigm which would utilize the innate neurophysiology of the human brain's speech system. In this experiment, two participants were asked to respond to a yes or no question via an EEG-based BCI of three different types; SSVEP-based, motor imagery-based, and laryngeal-imagery-based. By comparing the accuracy of the two established BCI paradigms to the novel laryngeal-imagery paradigm, we can establish the relative effectiveness of the novel paradigm. Machine learning algorithms were used to classify the EEG signals which had been transformed into frequency space (spectrograms) and common spatial pattern (CSP) dimensions. The SSVEP control task was able to be classified with better accuracy (62.5%) than the no information rate of 50% on the test set, but motor activity/imagery and laryngeal activity/imagery control tasks were not. Although the laryngeal methods did not produce accuracies above the no information rate, it is possible that with a larger amount of higher-quality data, this could prove otherwise. In the future, similar research should focus on reproducing the methods used here with better quality and more data.

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