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1.  Activation of Specific Apoptotic Caspases with an Engineered Small Molecule-Activated Protease 
Cell  2010;142(4):637-646.
Summary
Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the non-redundant roles of the executioner caspases-3, -6 and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-Tobacco Etch Virus (TEV) protease under small-molecule control, that we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.
doi:10.1016/j.cell.2010.07.014
PMCID: PMC3689538  PMID: 20723762
2.  Dual-mode laparoscopic fluorescence image-guided surgery using a single camera 
Biomedical Optics Express  2012;3(8):1880-1890.
Iatrogenic nerve damage is a leading cause of morbidity associated with many common surgical procedures. Complications arising from these injuries may result in loss of function and/or sensation, muscle atrophy, and chronic neuropathy. Fluorescence image-guided surgery offers a potential solution for avoiding intraoperative nerve damage by highlighting nerves that are otherwise difficult to visualize. In this work we present the development of a single camera, dual-mode laparoscope that provides near simultaneous display of white-light and fluorescence images of nerves. The capability of the instrumentation is demonstrated through imaging several types of in situ rat nerves via a nerve specific contrast agent. Full color white light and high brightness fluorescence images and video of nerves as small as 100 µm in diameter are presented.
doi:10.1364/BOE.3.001880
PMCID: PMC3409706  PMID: 22876351
(170.3880) Medical and biological imaging; (170.2150) Endoscopic imaging; (300.2530) Fluorescence, laser-induced
3.  In vivo imaging of microscopic structures in the rat retina 
Purpose
The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.
Methods
Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy.
Results
Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF.
Conclusions
With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution.
doi:10.1167/iovs.09-3675
PMCID: PMC2873188  PMID: 19578019
4.  Small Molecule Activators of a Proenzyme 
Science (New York, N.Y.)  2009;326(5954):853-858.
Virtually all of the 560 human proteases are stored as inactive proenyzmes and are strictly regulated. We report the identification and characterization of small molecules that directly activate the apoptotic proenzymes, procaspases-3 and -6. Surprisingly, these compounds induce autoproteolytic activation by stabilizing a conformation that is both more active and more susceptible to intermolecular proteolysis. These procaspase activators bypass the normal upstream proapoptotic signaling cascades and induce rapid apoptosis in a variety of cell lines. Systematic biochemical and biophysical analyses identified a cluster of mutations in procaspase-3 that resist small molecule activation both in vitro and in cells. Compounds that induce gain-of-function are rare and the activators reported here will enable direct control of the homodimeric executioner caspases in apoptosis and in cellular differentiation.
doi:10.1126/science.1177585
PMCID: PMC2886848  PMID: 19892984
5.  Inhibitor Hijacking of Akt Activation 
Nature chemical biology  2009;5(7):484-493.
The kinase Akt plays a central role as a regulator of multiple growth factor input signals, making it an attractive anti-cancer drug target. A-443654 is an ATP-competitive Akt inhibitor. Unexpectedly, treatment of cells with A-443654 causes paradoxical hyperphosphorylation of Akt at its two regulatory sites (Thr308 and Ser473). We explore whether inhibitor-induced hyperphosphorylation of Akt by A-443654 is a consequence of disrupted feedback regulation at a pathway level or whether it is a direct consequence of inhibitor binding to the ATP binding site of Akt. Catalytically inactive mutants of Akt reveal that binding of an inhibitor to the ATP site of Akt is sufficient to directly cause hyperphosphorylation of the kinase in the absence of any pathway feedback effects. We conclude that ATP-competitive Akt inhibitors impart regulatory phosphorylation of their target kinase Akt providing new insights into both natural regulation of Akt activation and Akt inhibitors entering the clinic.
doi:10.1038/nchembio.183
PMCID: PMC2783590  PMID: 19465931
6.  Light-Induced Retinal Changes Observed with High-Resolution Autofluorescence Imaging of the Retinal Pigment Epithelium 
Purpose
Autofluorescence fundus imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) allows for imaging of individual retinal pigment epithelial (RPE) cells in vivo. In this study, the potential of retinal damage was investigated by using radiant exposure levels that are 2 to 150 times those used for routine imaging.
Methods
Macaque retinas were imaged in vivo with a fluorescence AOSLO. The retina was exposed to 568- or 830-nm light for 15 minutes at various intensities over a square ½° per side. Pre-and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2° field. Long-term AOSLO imaging was performed intermittently from 5 to 165 days after exposure. Exposures delivered over a uniform field were also investigated.
Results
Exposures to 568-nm light caused an immediate decrease in autofluorescence of RPE cells. Follow-up imaging revealed either full recovery of autofluorescence or long-term damage in the RPE cells at the exposure. The outcomes of AOSLO exposures and uniform field exposures of equal average power were not significantly different. No effects from 830-nm exposures were observed.
Conclusions
The study revealed a novel change in RPE autofluorescence induced by 568-nm light exposure. Retinal damage occurred as a direct result of total average power, independent of the light-delivery method. Because the exposures were near or below permissible levels in laser safety standards, these results suggest that caution should be used with exposure of the retina to visible light and that the safety standards should be re-evaluated for these exposure conditions.
doi:10.1167/iovs.07-1430
PMCID: PMC2790526  PMID: 18408191
7.  In-vivo imaging of retinal nerve fiber layer vasculature: imaging - histology comparison 
BMC Ophthalmology  2009;9:9.
Background
Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed.
Methods
We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue.
Results
Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections.
Conclusion
This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.
doi:10.1186/1471-2415-9-9
PMCID: PMC2744910  PMID: 19698151
8.  Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents 
The Journal of Cell Biology  2008;183(1):101-116.
Although Akt is known as a survival kinase, inhibitors of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway do not always induce substantial apoptosis. We show that silencing Akt1 alone, or any combination of Akt isoforms, can suppress the growth of tumors established from phosphatase and tensin homologue–null human cancer cells. Although these findings indicate that Akt is essential for tumor maintenance, most tumors eventually rebound. Akt knockdown or inactivation with small molecule inhibitors did not induce significant apoptosis but rather markedly increased autophagy. Further treatment with the lysosomotropic agent chloroquine caused accumulation of abnormal autophagolysosomes and reactive oxygen species, leading to accelerated cell death in vitro and complete tumor remission in vivo. Cell death was also promoted when Akt inhibition was combined with the vacuolar H+–adenosine triphosphatase inhibitor bafilomycin A1 or with cathepsin inhibition. These results suggest that blocking lysosomal degradation can be detrimental to cancer cell survival when autophagy is activated, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PI3K–Akt pathway inhibition.
doi:10.1083/jcb.200801099
PMCID: PMC2557046  PMID: 18838554
9.  pHUSH: a single vector system for conditional gene expression 
BMC Biotechnology  2007;7:61.
Background
Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector.
Results
Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer.
Conclusion
We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.
doi:10.1186/1472-6750-7-61
PMCID: PMC2174931  PMID: 17897455

Results 1-9 (9)