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1.  Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes 
Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.
doi:10.1038/mtna.2013.25
PMCID: PMC3696905  PMID: 23778500
bioluminescence imaging; firefly luciferase; Gaussia luciferase; Vargula luciferase
2.  Lanatoside C sensitizes glioblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand and induces an alternative cell death pathway 
Neuro-Oncology  2011;13(11):1213-1224.
Human glioblastoma (GBM) cells are notorious for their resistance to apoptosis-inducing therapeutics. We have identified lanatoside C as a sensitizer of GBM cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced cell death partly by upregulation of the death receptor 5. We show that lanatoside C sensitizes GBM cells to TRAIL-induced apoptosis in a GBM xenograft model in vivo. Lanatoside C on its own serves as a therapeutic agent against GBM by activating a caspase-independent cell death pathway. Cells treated with lanatoside C showed necrotic cell morphology with absence of caspase activation, low mitochondrial membrane potential, and early intracellular ATP depletion. In conclusion, lanatoside C sensitizes GBM cells to TRAIL-induced cell death and mitigates apoptosis resistance of glioblastoma cells by inducing an alternative cell death pathway. To our knowledge, this is one of the first examples of use of caspase-independent cell death inducers to trigger tumor regression in vivo. Activation of such mechanism may be a useful strategy to counter resistance of cancer cells to apoptosis.
doi:10.1093/neuonc/nor067
PMCID: PMC3199161  PMID: 21757445
cardiac glycoside; glioblastoma; lanatoside C; non-apoptotic cell death; TRAIL
3.  Functional Drug Screening Assay Reveals Potential Glioma Therapeutics 
Abstract
Here we describe a novel functional screening assay based on bioluminescence monitoring of the naturally secreted Gaussia luciferase (Gluc) in the conditioned medium of cultured cells. Using this assay, we identified small-molecule drugs that sensitized brain tumor cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. Human glioblastoma multiforme cells were engineered by gene transfer to express Gluc as a reporter for cell viability, which can be monitored over time by bioluminescence measurements using a plate luminometer. We have optimized the Gluc assay for screening and validated it using the National Institute of Neurological Disorders and Stroke (NINDS) custom collection II library consisting of 1,040 drugs and bioactive compounds, most of which are Food and Drug Administration-approved and are able to cross the blood–brain barrier. We found that the cardiac glycosides family sensitized glioblastoma multiforme cells to the tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. In conclusion, the Gluc secretion assay is a robust tool for functional drug screening and can be applied to many different fields including cancer.
doi:10.1089/adt.2010.0324
PMCID: PMC3102258  PMID: 21184646
4.  A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress 
PLoS ONE  2007;2(6):e571.
Background
The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc).
Methodology/Principle Findings
An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells.
Conclusions/Significance
The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced.
doi:10.1371/journal.pone.0000571
PMCID: PMC1892804  PMID: 17593970

Results 1-4 (4)