Luciferase Bioluminescence Research Articles

Development of an Oxygen-Sensitive Degradable Peptide Probe for the Imaging of Hypoxia-Inducible Factor-1-Active Regions in Tumors

We aimed to develop a radiolabeled peptide probe for the imaging of hypoxia-inducible factor-1 (HIF-1)-active tumors. We synthesized the peptide probes that contain or lack an essential sequence of the oxygen-dependent degradation of HIF-1α in proteasomes ((123/125)I-DKOP30 or (125)I-mDKOP, respectively). The degradation of probes was evaluated in vitro using cell lysates containing proteasomes. In vivo biodistribution study, planar imaging, autoradiography, and comparison between probe accumulation and HIF-1 transcriptional activity were also performed. The (125)I-DKOP30 underwent degradation in a proteasome-dependent manner, while (125)I-mDKOP was not degraded. Biodistribution analysis showed (125)I-DKOP30 accumulation in tumors. The tumors were clearly visualized by in vivo imaging, and intratumoral distribution of (125)I-DKOP30 coincided with the HIF-1α-positive hypoxic regions. Tumoral accumulation of (125)I-DKOP30 was significantly correlated with HIF-1-dependent luciferase bioluminescence, while that of (125)I-mDKOP was not. (123)I-DKOP30 is a useful peptide probe for the imaging of HIF-1-active tumors.

Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin

PurposeTo assess P-glycoprotein (P-gp)-modulation ability and the mechanisms of P-gp inhibition mediated by a new synthetic rifampicin derivative, 1,8-dibenzoyl-rifampicin (DiBenzRif), in an in vitro model of the blood–brain barrier (BBB), RBE4 cells, and in membrane mimetic models (liposomes). MethodsP-gp expression (western blot) and activity [rhodamine 123 accumulation studies] were assessed until 72h of exposure to DiBenzRif. The effects on intracellular ATP levels and on P-gp ATPase activity were studied using luciferin–luciferase bioluminescence assay. Membrane fluidity changes were tracked by steady-state anisotropy measurements. Non-P-gp-related rhodamine 123 accumulation was evaluated using liposomes prepared with the main lipids present in RBE4 cell membranes. ResultsA significant increase in intracellular rhodamine 123 content was observed in DiBenzRif-treated cells at all tested time-points. This effect was associated with a significant reduction in ATP intracellular levels, the inhibition of P-gp ATPase activity and a significant increase in membrane fluidity. DiBenzRif also favoured rhodamine 123 accumulation in a liposomal model of RBE4 cells, suggesting that it may be useful in increasing intracellular levels of substances that passively diffuse into the cells. ConclusionDiBenzRif-induced inhibitory effect on P-gp increases xenobiotic accumulation in BBB cells, which may contribute to the development of therapeutic adjuvants to enhance brain penetration of drugs.

Abstract 3969: Blocking colony stimulating factor 1 receptor (CSF1R) improves anti-tumor effects of adoptive T cell transfer therapy.

Abstract Tumors can escape immune responses by creating an inflammatory milieu that supports and recruits tumor-infiltrating myeloid cells (TIMs) with immune suppressive functions, such as myeloid derived suppressor cells (MDSC) and M2-polarized macrophages. Therapies that block recruitment of suppressor TIMs have the potential to enhance adoptive cell transfer (ACT) of T cell based-immunotherapies. We originally established a BRAFV600E mutant murine melanoma cell line (SM1) for which ACT of melanoma-targeted T cells induced anti-tumor responses, but not complete eradication in vivo. By gene expression profiling and single-nucleotide polymorphism (SNP) arrays, we found that, out of 108 cytokines produced by SM1, colony stimulating factor 1 (CSF1) was highly expressed. CSF1 induces proliferation of immune suppressors Gr-1(+) CD11b(+) MDSC and F4/80(+) CD11b(+) macrophages. In order to block the recruitment of TIMs, we tested a potent CSF1R inhibitor, PLX3397. Cultured primary murine T cells exposed to PLX3397 with a broad range of concentrations (0.1 to 50 μM) showed no evidence of cytotoxicity by MTS assay. We then tested the combination of PLX3397 and ACT in vivo using two models. In the first one, SM1 cells stably expressing the chicken ovalbumin (OVA) antigen were implanted in C57BL/6 mice. Daily oral gavage with PLX3397 (50mg/kg) combined with ACT of OVA-specific TCR transgenic cells (OT-1) demonstrated superior effects of the combined treatment (tumor size on day 14_vehicle: 1611.2±22.5 mm2, PLX3397: 1028.0±24.0 mm2, OT-1: 1112.5±35.7 mm2, combined PLX3397+OT-1: 347.3±15.5 mm2, p<0.001). We have observed a dramatic reduction of the number of macrophages in the tumor tissue (vehicle: 10.3±1.6%, PLX3397: 1.35±0.5%, OT-1: 12.0±0.6%, combined: 2.33±0.3%, p<0.001) and a skewing of type M2 to type M1 macrophages. In vivo real-time T cell tracking by luciferase bioluminescence imaging demonstrated an increased number of tumor infiltrating lymphocytes (TILs) in the combined therapy group (photon counts/pixel on day 5 post ACT: OT-1: 4424.5±412, combined: 9176±1780, p<0.001). The second in vivo model was based on pmel-1 transgenic T cells, which targeted the endogenous melanoma antigen (gp100) expressed by SM1. The combined treatment of PLX3397 and ACT of pmel-1 also resulted in significant tumor shrinkage, reduction of M2 macrophages, and increased number of TILs. TILs were also collected and analyzed for function by intracellular staining of IFNγ. TILs from the combined treatment group released significantly higher levels of IFNγ (ACT: 48.2±8.7%, combined: 78.8±3.5%, p<0.001). In conclusion, our data derived from two independent in vivo models demonstrated dramatic potentiation of anti-tumor effects with the combination of CSF1R blockade and ACT immunotherapy, supporting the rationale for further testing in patients with metastatic melanoma. Citation Format: Stephen Mok, Christopher Tsui, Jingying Xu, Begonya Comin-Anduix, Thinle Chodon, Antoni Ribas, Richard C. Koya. Blocking colony stimulating factor 1 receptor (CSF1R) improves anti-tumor effects of adoptive T cell transfer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3969. doi:10.1158/1538-7445.AM2013-3969

Direct and Specific Effect of Sevoflurane Anesthesia on rat Per2 Expression in the Suprachiasmatic Nucleus

BackgroundOur previous studies revealed that application of the inhalation anesthetic, sevoflurane, reversibly repressed the expression of Per2 in the mouse suprachiasmatic nucleus (SCN). We aimed to examine whether sevoflurane directly affects the SCN.MethodsWe performed in vivo and in vitro experiments to investigate rat Per2 expression under sevoflurane-treatment. The in vivo effects of sevoflurane on rPer2 expression were examined by quantitative in situ hybridization with a radioactively-labeled cRNA probe. Additionally, we examined the effect of sevoflurane anesthesia on rest/activity rhythms in the rat. In the in vitro experiments, we applied sevoflurane to SCN explant cultures from Per2-dLuc transgenic rats, and monitored luciferase bioluminescence, representing Per2 promoter activity. Bioluminescence from two peripheral organs, the kidney cortex and the anterior pituitary gland, were also analyzed.ResultsApplication of sevoflurane in rats significantly suppressed Per2 expression in the SCN compared with untreated animals. We observed no sevoflurane-induced phase-shift in the rest/activity rhythms. In the in vitro experiments, the intermittent application of sevoflurane repressed the increase of Per2-dLuc luminescence and led to a phase delay in the Per2-dLuc luminescence rhythm. Sevoflurane treatment did not suppress bioluminescence in the kidney cortex or the anterior pituitary gland.ConclusionThe suppression of Per2-dLuc luminescence by sevoflurane in in vitro SCN cultures isolated from peripheral inputs and other nuclei suggest a direct action of sevoflurane on the SCN itself. That sevoflurane has no such effect on peripheral organs suggests that this action might be mediated through a neuron-specific cellular mechanism or a regulation of the signal transduction between neurons.

Simultaneous Measurement Device of the Luciferase Bioluminescence and the Delayed Fluorescence in Lettuce

Abstract In this study, we developed the devise named “Kondotron-lettuce” that is possible to measure luciferase bioluminescence and delayed fluorescence intensity of plants which are in the state that nearer to plant factory than the conventional circadian rhythm measurement. The main refinements are two points: One is that the devise became able to measure bioluminescence from plants which are cultured under hydroponic culturing for a long term. The another is that the devise became able to measure the delayed fluorescence. This devise is useful for measuring of luciferase bioluminescence and delayed fluorescence intensity under the controlled light and hydroponic culturing conditions in the long term.

Using C-Arm X-Ray Imaging to Guide Local Reporter Probe Delivery for Tracking Stem Cell Engraftment

Poor cell survival and difficulties with visualization of cell delivery are major problems with current cell transplantation methods. To protect cells from early destruction, microencapsulation methods have been developed. The addition of a contrast agent to the microcapsule also could enable tracking by MR, ultrasound, and X-ray imaging. However, determining the cell viability within the microcapsule still remains an issue. Reporter gene imaging provides a way to determine cell viability, but delivery of the reporter probe by systemic injection may be hindered in ischemic diseases.In the present study, mesenchymal stem cells (MSCs) were transfected with triple fusion reporter gene containing red fluorescent protein, truncated thymidine kinase (SPECT/PET reporter) and firefly luciferase (bioluminescence reporter). Transfected cells were microencapsulated in either unlabeled or perfluorooctylbromide (PFOB) impregnated alginate. The addition of PFOB provided radiopacity to enable visualization of the microcapsules by X-ray imaging. Before intramuscular transplantation in rabbit thigh muscle, the microcapsules were incubated with D-luciferin, and bioluminescence imaging (BLI) was performed immediately. Twenty-four and forty-eight hours post transplantation, c-arm CT was used to target the luciferin to the X-ray-visible microcapsules for BLI cell viability assessment, rather than systemic reporter probe injections. Not only was the bioluminescent signal emission from the PFOB-encapsulated MSCs confirmed as compared to non-encapsulated, naked MSCs, but over 90% of injection sites of PFOB-encapsulated MSCs were visible on c-arm CT. The latter aided in successful targeting of the reporter probe to injection sites using conventional X-ray imaging to determine cell viability at 1-2 days post transplantation. Blind luciferin injections to the approximate location of unlabeled microcapsules resulted in successful BLI signal detection in only 18% of injections. In conclusion, reporter gene probes can be more precisely targeted using c-arm CT for in vivo transplant viability assessment, thereby avoiding large and costly systemic injections of a reporter probe.

Near‐infrared fluorescence imaging of gastrin releasing peptide receptor targeting in prostate cancer lymph node metastases

Development of high affinity and specificity molecular imaging probes that increase accuracy for early detection of lymph node (LN) metastases is important for improving survivorship in prostate cancer. We evaluated the specificity, sensitivity, and accuracy of fluorescence-labeled bombesin (BBN) peptides to detect LN and systematic metastases in orthotopic mouse models bearing gastrin releasing peptide receptor (GRPR)-positive human prostate cancer. PC-3 cells were orthotopically implanted in severe combined immunedeficient or thymic nude (nu/nu) male mice. Tumor growth was monitored using magnetic resonance imaging. Alexa Fluor 680 conjugated BBN[7-14]NH2 (AF680-BBN) peptides were administered intravenously at 4-7 weeks post-tumor-implantation. Near-infrared fluorescence (NIRF) imaging was performed for up to 6 hr post-injection. The imaging sensitivity and specificity were assessed by co-registration of AF680-BBN NIRF imaging and luciferase bioluminescence imaging of the PC-3/Luc+ orthotopic mouse model. AF680-BBN showed a high binding affinity and selectivity to GRPR-positive cancer in vitro and in vivo. LN and peritoneal metastases were detected by NIRF imaging, and confirmed by histopathology. Tumor-to-muscle (T/M) ratio was the highest at 2-hr post-injection (4.12 ± 1.77). Blocking experiments, using unlabeled BBN as the inhibiting agent, significantly reduced the T/M ratio (1.64 ± 0.21, P = 0.02). AF680-BBN NIRF imaging had a sensitivity of 89.4%, specificity of 92.9%, and accuracy of 90.2% for the detection of metastases in mice. [corrected] The studies suggest the potential of use and development of NIR-fluorescent BBN probes as site-directed agents to help improve the current detection and LN staging accuracy in prostate cancer.

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

BackgroundCalcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.ResultsThe Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis.ConclusionsThis study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.

Low Molecular Weight Dextran Sulfate Binds to Human Myoblasts and Improves their Survival after Transplantation in Mice

Introduction: Myoblasts are skeletal muscle progenitors cells responsible for skeletal muscle growth and regeneration. As such and because myoblasts faces the complex challenges of clinical application, myoblast transplantation appears to be a promising approach for treatment of extensive skeletal muscle destruction occurring in severe myopathies such as Duchenne muscular dystrophy. Nevertheless, even in autologous combination, such approaches are impaired by the rapid death, limited migration and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant in vitro and in vivo. These beneficial effects of DXS in modulating innate immunity make this substance a possible candidate to improve the success of myoblast transplantation. Methods: Clonal cultures of human myoblasts were prepared from human muscle biopsies obtained during corrective orthopedic surgery. Myoblasts were 100% positives for the myogenic markers CD56 and desmin as assessed by flow cytometry. For in vivo experiments, human myoblasts were transduced with a lentiviral vector encoding the renilla luciferase bioluminescence marker gene (Rluc). Non-invasive bioluminescence imaging was used to quantify survival of engrafted human myoblasts after transplantation in immunodeficient mice. All research with human samples and mice was performed in compliance with the institutional guidelines, national as well as international guidelines. Results: In vitro, DXS binds dose dependently to human myoblasts and significantly inhibits staurosporine mediated apoptosis and necrosis. DXS pretreatment also protects human myoblasts from natural killer cell-mediated cytotoxicity in a dose dependent manner. At a multiplicity of infection of 1, 80% of the cells expressed the renilla luciferase transgene after lentiviral infection. Rluc transduced myoblasts kept their myogenic capacity to proliferate and to differentiate in vitro as assessed by fusion indexes. In vivo, we observed a two-fold improvement in survival of human myoblasts that were pretreated with DXS (10 mg/ml) as compared to untreated cells, at 1 day and at 3 days after transplantation (77.9 ± 10.1% vs 39.4 ± 4.9%; p = 0.0009 and 38.1 ± 8.5% vs 15.1 ± 3.4%; p = 0.01, respectively). No significant difference was observed at 7 days (8.0 ± 2.1 % vs 3.6 ± 0.7%; p = 0.06) and at 14 days post injection with values that were indistinguishable from background levels. Conclusion: Taken together, we provide evidence that DXS act as a myoblast protectant in vitro and in vivo, able locally to prevent early cell death. This protective effect of DXS was efficient up to 72h after pretreatment. DXS might represent a useful therapeutic reagent to improve the success of myoblast cell transplantation. More experiments are needed to evaluate if combination of DXS pretreatment of human myoblasts with systemic DXS administration of the recipient may be envisaged to enhance myoblast transplantation success.

Analysis of Biodistribution and Engraftment into the Liver of Genetically Modified Mesenchymal Stromal Cells Derived from Adipose Tissue

Presently, orthotopic liver transplant is the major therapeutic option for patients affected by primary liver diseases. This procedure is characterized by major invasive surgery, scarcity of donor organs, high costs, and lifelong immunosuppressive treatment. Transplant of hepatic precursor cells represents an attractive alternative. These cells could be used either for allogeneic transplantation or for autologous transplant after ex vivo genetic modification. We used stromal cells isolated from adipose tissue (AT-SCs) as platforms for autologous cell-mediated gene therapy. AT-SCs were transduced with lentiviral vectors expressing firefly luciferase, allowing for transplanted cell tracking by bioluminescent imaging (BLI). As a complementary approach, we followed circulating human α1-antitrypsin (hAAT) levels after infusion of AT-SCs overexpressing hAAT. Cells were transplanted into syngeneic mice after CCl(4)-induced hepatic injury. Luciferase bioluminescence signals and serum hAAT levels were measured at different time points after transplantation and demonstrate persistence of transplanted cells for up to 2 months after administration. These data, along with immunohistochemical analysis, suggest engraftment and repopulation of injured livers by transplanted AT-SCs. Moreover, by transcriptional targeting using cellular tissue-specific regulatory sequences, we confirmed that AT-SCs differentiate towards a hepatogenic-like phenotype in vitro and in vivo. Additionally, in transplanted cells reisolated from recipient animals' livers, we detected activation of the α-fetoprotein (AFP) promoter. This promoter is normally transcriptionally silenced in adult tissues but can be reactivated during liver regeneration, suggesting commitment towards hepatogenic-like differentiation of engrafted cells in vivo. Our data support AT-SC-mediated gene therapy as an innovative therapeutic option for disorders of liver metabolism.

Circadian rhythms of gastrointestinal function are regulated by both central and peripheral oscillators

Circadian clocks are responsible for daily rhythms in a wide array of processes, including gastrointestinal (GI) function. These are vital for normal digestive rhythms and overall health. Previous studies demonstrated circadian clocks within the cells of GI tissue. The present study examines the roles played by the suprachiasmatic nuclei (SCN), master circadian pacemaker for overt circadian rhythms, and the sympathetic nervous system in regulation of circadian GI rhythms in the mouse Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6-hydroxydopamine has no effect on feeding and locomotor rhythmicity in light-dark cycles or constant darkness but attenuates stool weight and stool number rhythms. Again, however, restricted feeding reestablishes rhythms in locomotor activity, feeding, and stool output rhythms. Ex vivo, intestinal tissue from PER2::LUC transgenic mice expresses circadian rhythms of luciferase bioluminescence. Chemical sympathectomy has little effect on these rhythms, but timed administration of the β-adrenergic agonist isoproterenol causes a phase-dependent shift in PERIOD2 expression rhythms. Collectively, the data suggest that the SCN are required to maintain feeding, locomotor, and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the GI system.

BioKnife, a uPA Activity-dependent Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma via Simultaneous Stimulation of uPA Expression

Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

An improved single-step lysis protocol to measure luciferase bioluminescence in Plasmodium falciparum

This report describes the optimization and evaluation of a simple single-step lysis protocol to measure luciferase bioluminescence from genetically modified Plasmodium falciparum. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. The utility of this improved assay protocol is demonstrated in typical assays that explore absolute and temporal gene expression activity.

TD-DFT/Molecular Mechanics Study of the Photinus pyralis Bioluminescence System

This is the first report of a computational study of the bioluminescence of ligand-bound Photinus pyralis luciferase. A time-dependent PBE0/molecular mechanics approach was used to study the interaction between excited-state oxyluciferin (Keto-(-1)) and neighboring active site molecules. The results of these calculations demonstrated that the most important intermolecular interactions are: blue-shifting ionic interactions, red-shifting π-π stacking, and red/blue shifting hydrogen bonding. Subsequent molecular dynamics simulations further supported these conclusions.

Evaluation of the Use of the TobaccoPR-1aPromoter to Monitor Defense Gene Expression by the Luciferase Bioluminescence Reporter System

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.

Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to solid tumors

Microbubble ultrasound contrast agents are being developed as image-guided gene carriers for targeted delivery in vivo. In this study, novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched polyethylenimine (PEI; 25kDa) was modified with polyethylene glycol (PEG; 5kDa), thiolated and covalently attached to maleimide groups on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content. The in vivo ultrasound contrast persistence of PEI-microbubbles was measured in the healthy mouse kidney, and a two-compartment pharmacokinetic model accounting for free and adherent microbubbles was developed to describe the anomalous time-intensity curves. The model suggested that PEI loading dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection.

Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells.

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.

A diversity of paracrine signals sustains molecular circadian cycling in suprachiasmatic nucleus circuits

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of mammals, coordinating daily rhythms of behavior and metabolism. Circadian timekeeping in SCN neurons revolves around transcriptional/posttranslational feedback loops, in which Period (Per) and Cryptochrome (Cry) genes are negatively regulated by their protein products. Recent studies have revealed, however, that these "core loops" also rely upon cytosolic and circuit-level properties for sustained oscillation. To characterize interneuronal signals responsible for robust pacemaking in SCN cells and circuits, we have developed a unique coculture technique using wild-type (WT) "graft" SCN to drive pacemaking (reported by PER2::LUCIFERASE bioluminescence) in "host" SCN deficient either in elements of neuropeptidergic signaling or in elements of the core feedback loop. We demonstrate that paracrine signaling is sufficient to restore cellular synchrony and amplitude of pacemaking in SCN circuits lacking vasoactive intestinal peptide (VIP). By using grafts with mutant circadian periods we show that pacemaking in the host SCN is specified by the genotype of the graft, confirming graft-derived factors as determinants of the host rhythm. By combining pharmacological with genetic manipulations, we show that a hierarchy of neuropeptidergic signals underpins this paracrine regulation, with a preeminent role for VIP augmented by contributions from arginine vasopressin (AVP) and gastrin-releasing peptide (GRP). Finally, we show that interneuronal signaling is sufficiently powerful to maintain circadian pacemaking in arrhythmic Cry-null SCN, deficient in essential elements of the transcriptional negative feedback loops. Thus, a hierarchy of paracrine neuropeptidergic signals determines cell- and circuit-level circadian pacemaking in the SCN.

Effects of Sucrose and Trehalose on Stability, Kinetic Properties, and Thermal Aggregation of Firefly Luciferase

In this study, we used sugars as stabilizing additives to improve the thermostability and to inhibit aggregation of firefly luciferase. The combination of sucrose and trehalose has a strong stabilizing effect on firefly luciferase activity and prevents its thermoinactivation. These additives can also increase optimum temperature. It has been shown that the presence of both sucrose and trehalose can inhibit thermal aggregation of firefly luciferase and decrease bioluminescence decay rate. In order to understand the molecular mechanism of thermostabilization, we investigated the effects of sucrose and trehalose combination on the secondary structure of luciferase by Fourier transform infrared spectroscopy. Minor changes in content of secondary structure of firefly luciferase are observed upon treatment with additives.