Role of self-compassion and online/offline integration on internet addiction, aggression, and psychological well-being: A mediation analysis.

Context: Internet addiction is known to harmfully affect psychological health. However, few researches have examined its plausible related factors and respite from its effects. Aim: This study aims to examine the relationship between internet addiction, aggression, psychological well-being, and the mediating effects of self-compassion and online/offline integration, on them. Materials and Methods: Data from 459 university students aged between 18 and 21 years were purposively selected from various disciplines and locations in India. Data were collected using an online demographic survey and standardized psychological measures. Statistical Analysis: Data analysis was performed using Partial Least Squares (PLS) path analysis. Direct and indirect effects and path coefficients were observed using PLS structural equation modeling. Results: The study indicated a possible influence of internet addiction on psychological well-being and aggression. It seems to increase aggression levels and lower psychological wellbeing. Online/offline integration and self-compassion partially mediated and dampened its adverse effects. Conclusions: Online/offline integration and self-compassion have a possibility to therapeutically diminish the ill-effects of internet addiction, lower aggression levels and promote psychological health of students who use internet extensively. This study provides a basis for further research to establish causal inferences.

Assessment of the validity of maturity metrics for predicting the volatile composition of Concord grape juice.

Total soluble solids (TSS) are typically used as the sole metric for maturity of Concord grapes. However, the reliability of TSS in predicting the aroma composition of finished juice for grapes sourced from multiple sites has not been investigated. This study sought to determine the validity of TSS in predicting the aroma composition of the juice while also investigating other potentially useful indicators, including pH, titratable acidity (TA), and TSS:TA ratio. Grapes were harvested from 9 sites in the Lake Erie Concord Grape Belt and stratified from north to south and east to west. The key aroma compounds methyl anthranilate ("grapey") and trans-2-hexenal ("herbaceous") were quantified along with several other odorants. It was observed that while TSS was a robust predictor of monomeric anthocyanin content across sites, it was a poor predictor of aroma compounds in the finished juice. Conversely, pH, TA, and TSS:TA ratio were all significantly correlated with methyl anthranilate and trans-2-hexenal concentrations (P < 0.05) in samples equal to or exceeding 15 Brix, the industry minimum for grape maturity. These results indicate that parameters related to acidity are better predictors of aroma composition than TSS, which may aid in efforts to minimize herbaceous off-aromas and optimize the aroma composition of the finished juice.

Optimal partitioning theory revisited: nonstructural carbohydrates dominate root mass responses to nitrogen.

Under optimal partitioning theory (OPT), plants preferentially allocate biomass to acquire the resource that most limits growth. Within this framework, higher root mass under low nutrients is often assumed to reflect an allocation response to build more absorptive surface. However, higher root mass also could result from increased storage of total nonstructural carbohydrates (TNC) without an increase in non-storage mass or root surface area. To test the relative contributions of TNC and non-storage mass as components of root mass responses to resources, we grew seedlings of seven northern hardwood tree species (black, red, and white oak, sugar and red maple, American beech, and black cherry) in a factorial light x nitrogen (N) greenhouse experiment. Because root mass is a coarse metric of absorptive surface, we also examined treatment effects on fine-root surface area (FRSA). Consistent with OPT, total root mass as a proportion of whole-plant mass generally was greater in low vs. high N. However, changes in root mass were influenced by TNC mass in all seven species and were especially strong in the three oak species. In contrast, non-storage mass contributed to increased total root mass under low N in three of the seven species. Root morphology also responded, with higher fine-root surface area (normalized to root mass) under low vs. high N in four species. Although biomass partitioning responses to resources were consistent with OPT, our results challenge the implicit assumption that increases in root mass under low nutrient levels primarily reflect allocation shifts to build more root surface area. Rather, root responses to low N included increases in: TNC, non-storage mass and fine-root surface area, with increases in TNC being the largest and most consistent of these responses. The greatest TNC accumulation occurred when C was abundant relative to N. Total nonstructural carbohydrates storage could provide seedlings a carbon buffer when respiratory or growth demands are not synchronized with photosynthesis, flexibility in responding to uncertain and fluctuating abiotic and biotic conditions, and increased access to soil resources by providing an energy source for mycorrhizae, decomposers in the rhizosphere, or root uptake of nutrients.

A pilot study to assess frequency of carriage and routes of acquisition of Staphylococcus aureus by healthy infants.

Healthy infants frequently acquire Staphylococcus aureus colonization; however, the modes of transmission are not well defined. In this study, 8 of 23 (35%) infants cultured at age 2 weeks acquired S aureus carriage, but only 1 infant had a family member with nasal carriage of the same clone, suggesting that sources other than colonized family members may account for a significant proportion of cases.

Imaging mitogen-activated protein kinase function in xenograft models of prostate cancer.

Mitogen-activated protein kinases (MAPK) play important roles in malignancy. The ability to detect and quantitate MAPKs in live animal models of cancer will facilitate an understanding of disease progression. We have developed a gene expression-based imaging system that detects and quantifies MAPK activity in prostate cancer tumors implanted into severe combined immunodeficient mice. The imaging technology uses a modified version of two-step transcriptional amplification (TSTA). The tissue specificity of gene expression is imparted by an enhanced version of the prostate-specific antigen regulatory region that expresses GAL4-ELK1. GAL4-ELK1 confers MAPK specificity by activating a firefly luciferase (FLuc) reporter gene when the Ets-like transcription factor (ELK) 1 activation domain is phosphorylated by MAPK. FLuc activity in live animals was detected using the Xenogen In vivo Imaging System. We validated the TSTA-ELK1 system by analyzing its response to epidermal growth factor treatment in transfected tissue culture cells and in adenovirus (AdTSTA-ELK1)-injected prostate cancer xenograft tumors. We measured MAPK activity in two well-characterized xenograft models, CWR22 and LAPC9. Although no significant differences in MAPK levels were detected between androgen-dependent and androgen-independent xenografts, the CWR22 models display significantly higher levels of AdTSTA-ELK1 activity versus LAPC9. Western blots of tumor extracts showed that the elevated imaging signal in CWR22 xenografts correlated with elevated levels of phosphorylated extracellular signal-regulated kinase 1/2 but not p38 or c-Jun NH(2)-terminal kinase. We conclude that a gene expression-based optical imaging system can accurately detect and quantify MAPK activity in live animals.

Bifunctional antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors.

An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.

Bioluminescence imaging of systemic tumor targeting using a prostate-specific lentiviral vector.

Developments in vector design using tissue-specific and tumor-specific promoters have led to significant improvements in tumor-targeting strategies. These developments combined with the ability to monitor gene expression by molecular imaging have facilitated the detection and prolonged monitoring of disease progression in small-animal models. Bioluminescence imaging offers a convenient and sensitive platform for monitoring gene expression patterns in preclinical models of gene therapy. Targeting a specific subset of cells/tissues via systemic delivery of vectors would be highly beneficial in gene therapy protocols. Using a two-step transcriptional amplification (TSTA)-based lentiviral vector (LV-TSTA), we demonstrate specific targeting of prostate tumors in vivo after systemic administration of lentivirus. Four days after intravenous administration of LV-TSTA into adult severe combined immunodeficient (SCID) mice (n=5) carrying subcutaneous prostate tumors, we found significant levels of transduction at the tumor site when compared with other organs (p<0.05). Gene expression was sustained in the tumor for up to 3 weeks (7.3x10(4)+/-2x10(4) photons/ sec/cm2/steradian (p/sec/cm2/sr) on day 4 and 7.0x10(4)+/-4x10(4) p/sec/cm2/sr on day 21). Low levels of transduction were also observed in the spleen and liver (5.0x10(2)+/-1.7x10(2) p/sec/cm2/sr). The results from this study support the use of TSTA-based lentiviral vectors for prostate tumor targeting after systemic delivery. Noninvasive imaging using such vectors should be useful for monitoring long-term gene expression in gene therapy applications.

Noninvasive indirect imaging of vascular endothelial growth factor gene expression using bioluminescence imaging in living transgenic mice.

Vascular endothelial growth factor (VEGF) plays a critical role in the early activation of stromal tissues during wound healing and tumor growth. We report the use of a two-step transcriptional amplification (TSTA) approach to augment the transcriptional activity of the relatively weak VEGF promoter (pVEGF) using firefly luciferase (fl) reporter gene and bioluminescence imaging (BLI). In cell culture, we demonstrate that TSTA-based fl gene expression can be significantly enhanced over the direct one-step system. Using a transgenic mouse model (pVEGF-TSTA-fl), we demonstrate the induction of VEGF gene expression using a wound-healing model and a subcutaneous mammary tumor model. In skin-wounding experiments, pVEGF-induced fl expression in the wound lesion is detected on days 4 and 5 and peaks on days 15-22. Furthermore, the bioluminescence signal shows good correlation with the endogenous VEGF protein levels in the wound tissue (r2 = 0.70). In the mammary tumor model, fl expression is detected on day 3, peaks at day 17, and declines thereafter. These results support the use of noninvasive BLI for the longitudinal monitoring of VEGF induction during wound healing and tumor progression, and this mouse model should find use in various applications in which it is important to noninvasively study VEGF gene expression.

Imaging androgen receptor function during flutamide treatment in the LAPC9 xenograft model.

The current understanding of the response of androgen receptor to pharmacologic inhibitors in prostate cancer is derived primarily from serum prostate-specific antigen (PSA) levels. In this study, we test whether a novel androgen receptor-specific molecular imaging system is able to detect the action of the antiandrogen flutamide on androgen receptor function in xenograft models of prostate cancer. Adenoviruses bearing an optical imaging cassette containing an androgen receptor-responsive two-step transcriptional amplification system were injected into androgen-dependent and hormone-refractory tumors of animals undergoing systemic time-controlled release of the antiandrogen flutamide. Imaging of tumors with a cooled charge-coupled device camera revealed that the response of AdTSTA to flutamide is more sensitive and robust than serum PSA measurements. Flutamide inhibits the androgen signaling pathway in androgen-dependent but not refractory tumors. Analysis of androgen receptor and RNA polymerase II binding to the endogenous PSA gene by chromatin immunoprecipitation revealed that flutamide treatment and androgen withdrawal have different molecular mechanisms. The application of imaging technology to study animal models of cancer provides mechanistic insight into antiandrogen targeting of androgen receptor during disease progression.

Noninvasive monitoring of target gene expression by imaging reporter gene expression in living animals using improved bicistronic vectors.

UNLABELLED: Indirect, noninvasive imaging of therapeutic gene expression based on levels of reporter gene expression is a powerful tool to devise improved therapeutic strategies in cancer gene therapy. The use of bicistronic vectors carrying internal ribosome entry sites (IRESs) allows the coexpression of multiple gene products from the same promoter but leads to considerable attenuation of the downstream gene. In this study, we describe the use of 10 linked copies of the Gtx (homeodomain protein) IRES (abbreviated as SIRES) in place of the encephalomyocarditis (EMCV) IRES in mediating downstream reporter gene expression in cell culture and in vivo. METHODS: We constructed several plasmid vectors carrying different upstream and downstream reporter genes (herpes simplex virus type I thymidine kinase [tk], firefly luciferase [fl], and Renilla luciferase [rl]) placed between EMCV IRES and SIRES segments. RL, FL, and TK enzyme activities in N2a, C6, and 293 cells transiently transfected with these vectors were found to be significantly higher for the SIRES vectors than for the EMCV IRES vectors. For in vivo experiments, 4 stably transfected N2a cell lines were implanted in nude mice. The mice were imaged for rl and fl gene expression using a charged-coupled device (CCD) camera. For bioluminescence and microPET imaging of downstream gene expression of fl and tk genes, respectively, mice carrying 4 stably transfected xenografts were imaged using the CCD camera and microPET. RESULTS: In cell culture, using rl as the upstream gene, we demonstrate that the expression of the downstream tk gene is 12-fold greater using SIRES when compared with EMCV IRES. Furthermore, the expression of the 2 genes was highly correlated in N2a cells. In vivo bioluminescence imaging using 4 stably transfected N2a cell lines revealed increasing levels of rl and fl gene expression. Bioluminescence and microPET, respectively, of fl and tk reporter gene expression in nude mice bearing N2a tumor xenografts showed the gene expression mediated by SIRES to be 4- and 8-fold higher, respectively, than EMCV IRES. CONCLUSION: These findings support the use of SIRES bicistronic vectors for a better assessment of therapeutic gene expression based on reporter gene expression in living subjects.

Applications of molecular imaging in cancer gene therapy.

Gene-based therapy is a promising and flexible therapeutic approach to manage diverse types of cancer. The lack of convincing therapeutic success of current gene therapy protocols in part, can be attributed to the inability to monitor gene expression at the targeted site in the living subject. Linking molecular imaging to gene therapy will enable real-time assessment of the therapeutic process and the refinement of treatment protocols. This review will cover two common imaging modalities, positron emission tomography (PET) and bioluminescence imaging (BLI), used in pre-clinical and clinical gene therapy applications. Strategies to develop more specific and robust cancer gene therapy and imaging approaches will be discussed. Coupling PET to gene therapy of cancer has already been implemented in several clinical studies. This approach would help to improve the efficacy and safety of future gene therapy clinical trials.

A tracer kinetic model for 18F-FHBG for quantitating herpes simplex virus type 1 thymidine kinase reporter gene expression in living animals using PET.

UNLABELLED: Reporter probe 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG) and reporter gene mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) have been used for imaging reporter gene expression with PET. Current methods for quantitating the images using the percentage injected dose per gram of tissue do not distinguish between the effects of probe transport and subsequent phosphorylation. We therefore investigated tracer kinetic models for 18F-FHBG dynamic microPET data and noninvasive methods for determining blood time-activity curves in an adenoviral gene delivery model in mice. METHODS: 18F-FHBG (approximately 7.4 MBq [approximately 200 microCi]) was injected into 4 mice; 18F-FHBG concentrations in plasma and whole blood were measured from mouse heart left ventricle (LV) direct sampling. Replication-incompetent adenovirus (0-2 x 10(9) plaque-forming units) with the E1 region deleted (n = 8) or replaced by HSV1-sr39tk (n = 18) was tail-vein injected into mice. Mice were dynamically scanned using microPET (approximately 7.4 MBq [approximately 200 microCi] 18F-FHBG) over 1 h; regions of interest were drawn on images of the heart and liver. Serial whole blood 18F-FHBG concentrations were measured in 6 of the mice by LV sampling, and 1 least-squares ratio of the heart image to the LV time-activity curve was calculated for all 6 mice. For 2 control mice and 9 mice expressing HSV1-sr39tk, heart image (input function) and liver image time-activity curves (tissue curves) were fit to 2- and 3-compartment models using Levenberg-Marquardt nonlinear regression. The models were compared using an F statistic. HSV1-sr39TK enzyme activity was determined from liver samples and compared with model parameter estimates. For another 3 control mice and 6 HSV1-sr39TK-positive mice, the model-predicted relative percentage of metabolites was compared with high-performance liquid chromatography analysis. RESULTS: The ratio of 18F-FHBG in plasma to whole blood was 0.84 +/- 0.05 (mean +/- SE) by 30 s after injection. The least-squares ratio of the heart image time-activity curve to the LV time-activity curve was 0.83 +/- 0.02, consistent with the recovery coefficient for the partial-volume effect (0.81) based on independent measures of heart geometry. A 3-compartment model best described 18F-FHBG kinetics in mice expressing HSV1-sr39tk in the liver; a 2-compartment model best described the kinetics in control mice. The 3-compartment model parameter, k3, correlated well with the HSV1-sr39TK enzyme activity (r2 = 0.88). CONCLUSION: 18F-FHBG equilibrates rapidly between plasma and whole blood in mice. Heart image time-activity curves corrected for partial-volume effects well approximate LV time-activity curves and can be used as input functions for 2- and 3-compartment models. The model parameter k3 from the 3-compartment model can be used as a noninvasive estimate for HSV1-sr39TK reporter protein activity and can predict the relative percentage of metabolites.

Noninvasive imaging of enhanced prostate-specific gene expression using a two-step transcriptional amplification-based lentivirus vector.

Noninvasive evaluation of gene transfer to specific cells or tissues will allow for long-term, repetitive monitoring of transgene expression. Tissue-specific promoters that restrict the expression of a transgene to tumor cells play a vital role in cancer gene therapy imaging. In this study, we have developed a third-generation HIV-1-based lentivirus vector carrying a prostate-specific promoter to monitor the long-term, sustained expression of the firefly luciferase (fl) reporter gene in living mice. The fl gene in the transcriptionally targeted vector is driven by an enhanced prostate-specific antigen promoter in a two-step transcriptional amplification (TSTA) system. The efficiency of the lentivirus (LV-TSTA)-mediated gene delivery, cell-type specificity, and persistence of gene expression were evaluated in cell culture and in living mice carrying prostate tumor xenografts. In vivo bioluminescence imaging with a cooled charge-coupled device camera revealed significantly high levels of fl expression in prostate tumors. Injection of LV-TSTA directly into the prostate of male nude mice revealed efficient and long-term fl gene expression in the prostate tissue for up to 3 months. These studies demonstrate the significant potential of TSTA-based lentivirus vectors to confer high levels of tissue-specific gene expression from a weak promoter, while preserving cell-type specificity and the ability to image noninvasively the sustained, long-term expression of reporter genes in living animals.

Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression.

Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.

Comparison of [18F]FHBG and [14C]FIAU for imaging of HSV1-tk reporter gene expression: adenoviral infection vs stable transfection.

Earlier studies involving comparison of different reporter probes have shown conflicting results between pyrimidine nucleosides [e.g., 2'-fluoro-2'-deoxy-1-beta- d-arabinofuranosyl-5-iodouracil (FIAU)] and acycloguanosine derivatives [e.g., penciclovir (PCV), 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG)]. We hypothesized that this reported discrepancy may be related to how the reporter gene is delivered to the cells-stably transfected vs adenoviral infection. We directly compared the uptake characteristics of [(18)F]FHBG, [(3)H]PCV, and [(14)C]FIAU in cell culture and in vivo using an adenoviral mediated gene transfer model and stably transfected cells. We further compared the uptake of three reporter probes using both HSV1-tk and a mutant HSV1-sr39tk expressing cells to assess the optimal reporter probe/reporter gene combination. [(14)C]FIAU accumulation was greater than that of [(3)H]PCV and [(18)F]FHBG in control cells and in HSV1-tk stably transfected cells ( P<0.001). After infection of C6 cells with AdCMV- HSV1-tk (1.5x10(8) pfu), [(18)F]FHBG and [(3)H]PCV accumulation was significantly greater than that of [(14)C]FIAU ( P<0.01). [(18)F]FHBG and [(3)H]PCV accumulated to a significantly greater extent than [(14)C]FIAU in C6-stb-sr39tk+ and AdCMV- HSV1-sr39tk infected C6 cells ( P<0.001). Results from the nude mice supported the results in cell culture. [(14)C]FIAU led to significantly higher %ID/g in C6-stb-tk+ xenografts than [(18)F]FHBG ( P<0.05); however, compared with [(14)C]FIAU, [(18)F]FHBG led to as high %ID/g in HSV1-tk expressing hepatocytes and to significantly greater %ID/g in C6-stb-sr39tk+ xenografts and HSV1-sr39tk expressing hepatocytes. Dynamic sequential images showed that [(18)F]FHBG was well retained in HSV1-sr39tk expressing cells (C6-stb-sr39tk+) for at least 4 h after injection, while it was rapidly cleared from HSV1-tk expressing cells (MH3924A-stb-tk+). [(14)C]FIAU accumulated in HSV1-tk stably expressing cells to a greater extent than either [(3)H]PCV or [(18)F]FHBG. However, the accumulation of [(3)H]PCV and [(18)F]FHBG in adenoviral infected C6 cells or hepatocytes was equivalent to or greater than that of [(14)C]FIAU. These results may be due to intracellular biochemical changes (e.g., thymidine) when cells are infected with adenovirus. For adenoviral studies, the [(18)F]FHBG/ HSV1-sr39tk combination was shown to be more sensitive than the [(14)C]FIAU/ HSV1-tk combination HSV1-tk.

Interrogating androgen receptor function in recurrent prostate cancer.

The early androgen-dependent (AD) phase of prostate cancer is dependent on the androgen receptor (AR). However, it is unclear whether AR is fully functional in recurrent prostate cancer after androgen withdrawal. To address this issue we interrogated AR signaling in AD and recurrent prostate cancer xenografts using molecular imaging, chromatin immunoprecipitation, and immunohistochemistry. In the imaging experiments, an adenovirus bearing a two-step transcriptional activation cassette, which amplifies AR-dependent firefly luciferase reporter gene activity, was injected into tumors implanted into severe combined immunodeficiency mice. A charge-coupled device optical imaging system detected the initial loss and then resumption of AR transcriptional activity in D-luciferin-injected mice as tumors transitioned from AD to recurrent growth. The results of chromatin immunoprecipitation and immunohistochemical localization experiments correlated with the Ad two-step transcriptional activation imaging signal. AR localized to the nucleus and bound to the endogenous prostate-specific antigen enhancer in AD tumors but exited the nucleus and dissociated from the enhancer upon castration. However, AR reentered the nucleus and rebound the prostate-specific antigen enhancer as the cancer transitioned into the recurrent phase. Surprisingly, RNA polymerase II and the general factor TFIIB remained bound to the gene throughout the transition. Our data support the concept that AR is fully functional in recurrent cancer and suggest a model by which a poised but largely inactive transcription complex facilitates reactivation by AR at castrate levels of ligand.

Noninvasive imaging of cationic lipid-mediated delivery of optical and PET reporter genes in living mice.

Gene therapy involves the safe and effective delivery of one or more genes of interest to target cells in vivo. The advantages of using nonviral delivery systems include ease of preparation, low toxicity, and weak immunogenicity. Nonviral delivery methods, when combined with a noninvasive, clinically applicable imaging assay, will greatly aid in the optimization of gene therapy approaches for cancer. We demonstrate cationic lipid-mediated noninvasive monitoring of reporter gene expression of firefly (Photinus pyralis) luciferase (fl) and a mutant herpes simplex virus type I thymidine kinase (HSV1-sr39tk, tk) in living mice using a cooled charge coupled device (CCD) camera and positron emission tomography (PET), respectively. We observe a high level of fl and tk reporter gene expression predominantly in the lungs after a single injection of the extruded DOTAP:cholesterol DNA liposome complexes by way of the tail vein, seen to be time- and dose-dependent. We observe a good correlation between the in vivo bioluminescent signal and the ex vivo firefly luciferase enzyme (FL) activity in different organs. We further demonstrate the feasibility of noninvasively imaging both optical and PET reporter gene expression in the same animal using the CCD camera and microPET, respectively.

Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer.

Gene therapy is founded on the concept that tissue-specific promoters can express heterologous genes for molecular imaging or therapeutic applications. The engineering of cell-specific enhancers to improve potency and the development of two-step transcriptional activation (TSTA) approaches have significantly improved the efficacy of transgene expression. Here we combine these technologies to create a robust, titratable, androgen-responsive system targeted to prostate cancer cells. Our "chimeric" TSTA system uses a duplicated variant of the prostate-specific antigen (PSA) gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains. We targeted the resulting activators to cells with reporter templates bearing one, two, or five GAL4 binding sites upstream of firefly luciferase. We monitored activity via firefly luciferase assays in transfected cell extracts and in live nude mice using a cooled charge-coupled device (CCD) imaging system. In this system, we found that firefly luciferase expression in prostate cancer cells can be varied over an 800-fold range. We also found that a single plasmid bearing the optimized enhancer, GAL4-VP16 derivative, and reporter expressed firefly luciferase at 20-fold higher levels than the cytomegalovirus enhancer. We discuss the implications of this strategy and its application to molecular imaging and therapy.