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Pompe disease is caused by mutations in acid alpha glucosidase (GAA) that causes accumulation of lysosomal glycogen affecting the heart and skeletal muscles, and can be fatal. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) improves muscle function by reducing glycogen accumulation. Limitations of ERT include a short half-life and the formation of antibodies that result in reduced efficacy. By harnessing the immune tolerance induction properties of the liver, liver-targeted gene delivery (with an adeno-associated virus vector containing a liver specific promoter), suppresses immunity against the GAA introduced by gene therapy. This induces immune tolerance to rhGAA by activating regulatory T cells and simultaneously, corrects GAA deficiency. Potentially, liver-targeted gene therapy can be performed once with lasting effects, by administering a relatively low dose of an adeno-associated virus type 8 vector to replace and induce immune tolerance to GAA.

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Anadenobolus excisus is a large species of millipede endemic to the Caribbean Island of Jamaica. Initial detailed morphological studies showed little or no discrete variation across this species' distribution in somatic or, in particular. genitalic morphology. However, a molecular survey based on approximately 1000 base pairs of the mitochondrial (mtDNA) 16S rRNA gene that examines 242 individuals sampled from 54 localities reveals three highly divergent mtDNA lineages. A lack of discrete morphological differentiation suggests that genetic and morphological divergence may be decoupled, a pattern inconsistent with a number of evolutionary models. In contrast to minimal morphological divergence, size variation among mtDNA lineages suggests that character displacement has occurred and that these lineages are cohesive in sympatry. We conclude that A. excisus is actually a complex of three cryptic species and that morphological approaches to delineating millipede species may sometimes underestimate evolutionary diversity.

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Aptostichus simus is a trapdoor spider endemic to the coastal dunes of central and southern California and, on morphological grounds, is recognized as a single species. Mitochondrial DNA 16S rRNA sequences demonstrate that most populations are fixed for the same haplotype and that the population haplotypes from San Diego County, Los Angeles County, Santa Rosa Island, and Monterey County are extremely divergent (6-12%), with estimated separation times ranging from 2 to 6 million years. A statistical cluster analysis of morphological features demonstrates that this genetic divergence is not reflected in anatomical features that might signify ecological differentiation among these lineages. The species status of these divergent populations of A. simus depends upon the species concept utilized. If a time-limited genealogical perspective is employed, A. simus would be separated at the base into two genetically distinct species. This study suggests that species concepts based on morphological distinctiveness, in spider groups with limited dispersal capabilities, probably underestimate true evolutionary diversity.

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The purpose of this study was to develop techniques for registering image sets associated with staged or multifraction radiosurgical treatments of large targets with the Leksell gamma knife to transform shot coordinates between treatment sessions and produce cumulative dose distributions and to investigate the theoretical biological effects of such protracted treatments by means of such concepts as the linear-quadratic model and biologically effective dose. An image registration technique based on normalized mutual information was adapted to produce one fused-image study from an imaging series acquired during distinct treatment sessions. A spreadsheet computer program was developed to determine coordinate transformations between the associated stereotactic coordinate systems based on digitized coordinates of fiducial markers appearing on the fused images. Coordinates of shots used during one treatment session could then be transformed to the stereotactic space of another session, and cumulative dose distributions could be computed. The procedure was applied to the two-stage treatment of a giant arteriovenous malformation (AVM). Overall uncertainty in each transformed shot position is approximately 0.7 mm. An effective single-fraction dose (D(eff)) was defined and computed for the two-stage AVM treatment. The simple summed dose distribution was compared with the D(eff) distribution. Because dose values differ significantly in overlap regions between the individual distributions, the clinical usefulness of the simple cumulative distribution is dubious. It may be useful for a future update of the GammaPlan treatment planning software to generate effective single-session dose distributions for such cases.

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Various techniques have been developed to localize radioactive sources in brachytherapy implants. The most common methods include the orthogonal film method, the stereo-shift film method, and recently, direct localization from a series of contiguous CT transverse images. The major advantage of the CT method is that it provides the seed locations relative to anatomic structures. However, it is often the case that accurate identification and localization of the sources become difficult because of partial source artifacts in more than one transverse cut and other artifacts on CT images. A new algorithm has been developed to combine the advantages of using a pair of orthogonal scout views with the advantages of using a stack of transverse cuts. In the new algorithm, a common reference point is used to correlate CT transverse images and two orthogonal scout CT scans (AP and lateral). The radioactive sources are localized on CT transverse images. At the same time, the sources are displayed automatically on the two CT scout scans. In this way, the individual sources can be clearly distinguished and ambiguities arising from partial source artifacts are resolved immediately. Because of the finite slice thickness of transverse cuts, the longitudinal coordinates are more accurately obtained from the scout views. Therefore, the longitudinal coordinates of seeds localized on the transverse cuts are adjusted so that they match the position of the seeds on scout views. The algorithm has been tested on clinical cases and has proved to be a time saving and accurate method.

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Transgenic Washington navel orange [Citrus sinensis (L.) Osbeck] plants were obtained using Agrobacterium-mediated transformation of seedling epicotyl tissue. An average of 45% (58 out of 128 segments) of the epicotyl segments produced shoots expressing the ß-glucuronidase (GUS)-intron reporter gene when using Agrobacterium strain C58 C1, compared to 29% (38 out of 128 segments) for EHA101-5 and 0% for LBA4404. Co-culture of 21-day-old Washington navel epicotyl stem segments gave greater transformation efficiency than co-culture of 35- or 56-day-old stem segments. After 6 weeks, regenerated shoots were micro-grafted in vivo onto seedling rootstocks of Carrizo citrange. Stable integration of the transgene sequence was confirmed by expression of the plant intron-containing GUS gene, PCR and Southern hybridization. The apomictic (non-zygotic) state of the transgenic plants was confirmed by isoenzyme and random amplified polymorphic DNA analyses. More than 50 transgenic plants have been obtained and are growing in the greenhouse.

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The treatment plans for stereotactic radiosurgery employ small, circular, noncoplanar fields applied in a series of arcs, or with synchronous rotation of the accelerator gantry and patient support assembly. Primary or metastatic brain tumors and arterial-venous malformations are localized in relation to a stereotactic head frame using CT, MRI, and angiography. As x-ray doses in the range of 20-40 Gy are delivered in a single treatment, it is critical that the dose distribution produced by the accelerator accurately reflect the one developed by the treatment planning computer. Until the advent of Fricke-infused gels, whose NMR characteristics are changed by irradiation, there was no practical method for assessing the accuracy of x-ray beam positioning on a target that was localized by both CT and MRI. A stereotactic head frame was attached to a hollow glass head filled with a Fricke-infused gel. A 2-mm target point at approximately the center of this manikin was localized by CT and MRI. The head frame was then mounted to the patient support assembly of a linear accelerator, and given a dose of 40 Gy to the isocenter from 6-MV x rays using a modified version of the dynamic stereotactic radiosurgery plan developed in Montreal. Subsequent MRI showed the target point at the center of the dose distribution, thus confirming the accuracy of the stereotactic radiosurgery procedure. This demonstrated the unique characteristics of the Fricke-infused gel for the simultaneous localization of x-ray beams in three dimensions.

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The application of cavity-ionization chambers to the standadization of 60Co gamma-ray beams, in terms of exposure, requires that the specific ionization of air Jg, be corrected for the attenuation and scatter of the incident rays by the wall, central electrode, and supporting stem of the chamber. A Monte Carlo photon-electron transport code has been developed for the purpose of calculating this correction for spherical and cylindrical chambers. The code has been applied to a spherical graphite chamber having dimensions typical of the chambers used by the NBS, the calculated wall-correction factor is in close agreement with the average of the NBS factors which were determined experimentally. The code was also used to calculate Aeq, which is central to the determination of tissue-air ratios. The calculated value, 0.989 +/- 0.003, is very close to the generally accepted value, 0.985.

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