Faculty Bibliography

The expansion of gene therapy applications from inherited disorders to acquired conditions has been mirrored by an exponential rise in both experimental work and clinical trials. This review highlights current plastic surgical delivery systems and clinical applications for targeted gene therapy. We revisit some of the vectors used both experimentally and in clinical gene therapy trials, with an emphasis on developments in plastic surgical delivery systems resulting in improved targeting of therapeutic genes. In addition, we discuss a novel technique for the delivery of gene therapy using the ex vivo transduction of free flaps, developed in our laboratory. This delivery system achieves targeted high-level transgene expression with minimal demonstrable systemic toxicity. Advances in delivery systems are essential for translating basic research into clinical therapeutics

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development

Human exposure to air pollutants, including ambient particulate matter, has been proposed as a mechanism for the rise in allergic disorders. Diesel exhaust particles, a major component of ambient particulate matter, induce sensitization to neoallergens, but the mechanisms by which sensitization occur remain unclear. We show that diesel exhaust particles upregulate thymic stromal lymphopoietin in human bronchial epithelial cells in an oxidant-dependent manner. Thymic stromal lymphopoietin induced by diesel exhaust particles was associated with maturation of myeloid dendritic cells, which was blocked by anti-thymic stromal lymphopoietin antibodies or silencing epithelial cell-derived thymic stromal lymphopoietin. Dendritic cells exposed to diesel exhaust particle-treated human bronchial epithelial cells induced Th2 polarization in a thymic stromal lymphopoietin-dependent manner. These findings provide new insight into the mechanisms by which diesel exhaust particles modify human lung mucosal immunity

The adaptor protein SLP76 directs signaling downstream of the T cell receptor (TCR) and is essential for thymocyte development. SLP76 contains three N-terminal tyrosines that are critical for its function. To define the role of these residues in thymocyte development, we generated two lines of "knock-in" mice, one expressing a mutation in tyrosine 145 (Y145F) and a second harboring two point mutations at tyrosines 112 and 128 (Y112-128F). We show here that although thymocyte development requires both Y145- and Y112-128-generated signals, selection was more dependent upon Y145. Although several proximal TCR signaling events were defective in both mutant mice, phosphorylation of the guanine nucleotide exchange factor, Vav1, and activation of Itk-dependent pathways were differentially affected by mutations at Y112-128 and Y145, respectively. Analysis of mice expressing one Y145F and one Y112-128F allele revealed that these mutants could complement one another in trans, demonstrating cooperativity between two or more SLP76 molecules. Thus, the N-terminal tyrosines of SLP76 are required for thymocyte selection but can function on separate molecules to support TCR signaling.

Carbon dioxide (CO2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO2 receptors and form connections characteristic of CO2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO2

Germ cells are the only cell type capable of generating an entirely new organism. In order to execute germline-specific functions and to retain the capacity for totipotency, germ cells repress somatic differentiation, interact with a specialized microenvironment, and use germline-specific networks of RNA regulation

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% alpha-helix, 23% beta-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.

Clostridial botulinum neurotoxin (BoNT) causes a neuroparalytic condition recognized as botulism by arresting synaptic vesicle exocytosis. Although the crystal structures of full-length BoNT/A and BoNT/B holotoxins are known, the molecular architecture of the five other serotypes remains elusive. Here, we present the structures of BoNT/A and BoNT/E using single particle electron microscopy. Labeling of the particles with three different monoclonal antibodies raised against BoNT/E revealed the positions of their epitopes in the electron microscopy structure, thereby identifying the three hallmark domains of BoNT (protease, translocation, and receptor binding). Correspondingly, these antibodies selectively inhibit BoNT translocation activity as detected using a single molecule assay. The global structure of BoNT/E is strikingly different from that of BoNT/A despite strong sequence similarity. We postulate that the unique architecture of functionally conserved modules underlies the distinguishing attributes of BoNT/E and contributes to differences with BoNT/A.