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Sample records for activity luciferase reporter

  1. A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

    PubMed

    Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

    2016-01-01

    Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase. PMID:27316998

  2. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters

    PubMed Central

    Buckley, Suzanne M. K.; Delhove, Juliette M. K. M.; Perocheau, Dany P.; Karda, Rajvinder; Rahim, Ahad A.; Howe, Steven J.; Ward, Natalie J.; Birrell, Mark A.; Belvisi, Maria G.; Arbuthnot, Patrick; Johnson, Mark R.; Waddington, Simon N.; McKay, Tristan R.

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4−/− mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively. PMID:26138224

  3. Luciferase as a reporter of gene activity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since their development and introduction in the early days of plant genetic engineering, reporter genes have established a proven track record as effective tools for exploring the molecular underpinnings of gene regulation. When driven by appropriate genetic control systems (e.g. transcriptional pr...

  4. Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in rhipicephalus (boophilus) microplus cell lines.

    PubMed

    Tuckow, A P; Temeyer, K B

    2015-08-01

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vector of bovine babesiosis and anaplasmosis. Commercial promoters were evaluated for transcriptional activity driving luciferase expression in the tick cell lines. The human phosphoglycerate kinase (PGK) promoter resulted in detectable firefly luciferase activity within 2 days post-transfection of the R. microplus cell line BME26, with maximal activity at 5 days post-transfection. Several other promoters were weaker or inactive in the tick cells, prompting identification and assessment of transcriptional activity of the homologous ribosomal protein L4 (rpL4, GenBank accession no.: KM516205) and elongation factor 1α (EF-1α, GenBank accession no.: KM516204) promoters cloned from R. microplus. Evaluation of luciferase expression driven by various promoters in tick cell culture resulted in selection of the R. microplus rpL4 promoter and the human PGK promoter driving transcription of sequences encoding modified firefly and NanoLuc® luciferases for construction of a dual luciferase reporter system for use in tick cell culture. PMID:25892533

  5. Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity

    PubMed Central

    Kashima, Hajime; Momose, Fumiyasu; Umehara, Hiroshi; Miyoshi, Nao; Ogo, Naohisa; Muraoka, Daisuke; Shiku, Hiroshi; Harada, Naozumi; Asai, Akira

    2016-01-01

    Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors. PMID:27284967

  6. Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity

    PubMed Central

    Akhmedov, Dmitry; Rajendran, Kavitha; Mendoza-Rodriguez, Maria G.

    2016-01-01

    The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo. PMID:27336479

  7. Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators.

    PubMed

    Smirnova, Natalya A; Haskew-Layton, Renee E; Basso, Manuela; Hushpulian, Dmitry M; Payappilly, Jimmy B; Speer, Rachel E; Ahn, Young-Hoon; Rakhman, Ilay; Cole, Philip A; Pinto, John T; Ratan, Rajiv R; Gazaryan, Irina G

    2011-06-24

    The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found--nordihydroguaiaretic acid, fisetin, and gedunin--induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism. PMID:21700211

  8. Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators

    PubMed Central

    Smirnova, Natalya A.; Haskew-Layton, Renee E.; Basso, Manuela; Hushpulian, Dmitry M.; Payappilly, Jimmy B.; Speer, Rachel E.; Ahn, Young-Hoon; Rakhman, Ilay; Cole, Philip A.; Pinto, John T.; Ratan, Rajiv R.; Gazaryan, Irina G.

    2011-01-01

    SUMMARY The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin—induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism. PMID:21700211

  9. Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.

    PubMed

    Boulling, Arnaud; Escher, Pascal

    2016-07-01

    Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina. PMID:27268947

  10. A dioxin response element in the multiple cloning site of the pGL3 luciferase reporter influences transcriptional activity1

    PubMed Central

    Ochs, Sharon; Liu, Jing; Fernando, Tharu; Fecher, Roger; Sulentic, Courtney E.W.

    2012-01-01

    Luciferase reporter plasmids (pGL3 backbone, Promega) have been utilized to characterize the transcriptional effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands. Following ligand activation, the AhR and its dimerization partner AhR nuclear translocator (ARNT) regulate transcription by binding dioxin response elements (DREs) in regulatory regions of dioxin-sensitive genes. Upon sequencing of our luciferase reporters, we unexpectedly identified a DRE core motif within the multiple cloning site (mcsDRE) of the pGL3 luciferase plasmid backbone in a subset of our reporters. Therefore, the objective of this study was to determine if the mcsDRE inadvertently influences reporter activity. Utilizing deletional analysis we determined that the mcsDRE did significantly alter the transcriptional effect induced by TCDD. Since many chemicals have been shown to interact with the AhR and influence transcription through the DRE, the presence of the mcsDRE in the pGL3 luciferase plasmid may inappropriately influence promoter and enhancer analysis. As such, insertion of regulatory elements into pGL3 reporters should be designed to avoid retaining the mcsDRE core motif (GCGTG) and currently utilized pGL3 reporters should be evaluated for the presence of the mcsDRE. PMID:22652426

  11. A novel luciferase based reporter system to monitor activation of the ErbB2/Her2/neu pathway non-invasively during radiotherapy

    PubMed Central

    Wolf, Frank; Li, Wenrong; Li, Fang; Li, Chuan-Yuan

    2010-01-01

    Purpose To develop a split-luciferase based reporter system that allows for non-invasive monitoring of activation of the Her2/neu pathway in vivo in a quantitative and sensitive manner. Methods and Materials Fusion proteins of the ErbB2/Her2/neu receptor to the N-terminal fragment of luciferase as well as of its downstream binding partner Shc to the C-terminal fragment of luciferase have been engineered based on the rationale that upon activation and binding of the Her2 receptor molecule to Shc, luciferase function will be reconstituted. Thus the resulting bioluminescence signals can serve as a surrogate measure of receptor activation. Results We show that our reporter systems functions well in vitro in breast cancer cells and in vivo in xenograft tumors. In particular, the activities of Her2/neu in xenograft tumors could be monitored serially for an extended period of time after radiotherapy. Conclusions We believe that the novel ErbB2/Her2/neu reporter presented here is a powerful tool to study the biology of the Her2-neu pathway in vitro as well as in vivo. It should also facilitate the development and rapid evaluation of new Her2/neu targeted therapeutics. PMID:20934271

  12. Novel Luciferase-Based Reporter System to Monitor Activation of ErbB2/Her2/neu Pathway Noninvasively During Radiotherapy

    SciTech Connect

    Wolf, Frank; Li Wenrong; Li Fang; Li Chuanyuan

    2011-01-01

    Purpose: To develop a split-luciferase-based reporter system that allows for noninvasive monitoring of activation of the Her2/neu pathway in vivo in a quantitative and sensitive manner. Methods and Materials: Fusion proteins of the ErbB2/Her2/neu receptor to the N-terminal fragment of luciferase and of its downstream binding partner Shc to the C-terminal fragment of luciferase have been engineered owing to the rationale that on activation and binding of the Her2 receptor molecule to Shc, luciferase function will be reconstituted. Thus, the resulting bioluminescence signals can serve as a surrogate measure of receptor activation. Results: We have shown that our reporter systems functions well in vitro in breast cancer cells and in vivo in xenograft tumors. In particular, the activities of Her2/neu in xenograft tumors could be monitored serially for an extended period after radiotherapy. Conclusions: We believe that the novel ErbB2/Her2/neu reporter we have presented is a powerful tool to study the biology of the Her2-neu pathway in vitro and in vivo. It should also facilitate the development and rapid evaluation of new Her2/neu-targeted therapeutic agents.

  13. Discovery, adaptation and transcriptional activity of two tick promoters: Construction of a dual luciferase reporter system for optimization of RNA interference in Rhipicephalus (Boophilus) microplus cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vec...

  14. Bortezomib and ixazomib protect firefly luciferase from degradation and can flaw respective reporter gene assays.

    PubMed

    Becker, Jonas Philipp; Clemens, Jannick Robert; Theile, Dirk; Weiss, Johanna

    2016-09-15

    Firefly luciferase-based reporter gene assays are the most commonly used assays to investigate the transcriptional regulation of gene expression. However, direct interaction of tested compounds with the firefly luciferase leading to altered enzymatic activity may lead to misinterpretation of experimental data. When investigating the proteasome inhibitors bortezomib, carfilzomib, and ixazomib, we observed increased luminescence for bortezomib and ixazomib, but not for carfilzomib, in a pregnane-X-receptor (PXR) reporter gene assay, which was inconsistent with the mRNA expression levels of the main PXR target gene CYP3A4. To further scrutinize this phenomenon, we performed experiments with constitutively expressed firefly luciferase and demonstrated that the increase in cellular firefly luciferase activity is independent from PXR activation or CYP3A4 promoter. Using cell-free assays with recombinant firefly luciferase enzyme, we made the counterintuitive observation that firefly luciferase activity is inhibited by bortezomib and ixazomib in a reversible and competitive manner. This inhibition stabilizes the firefly luciferase enzyme against proteolytic degradation (e.g., toward trypsin), thereby increasing its half-life with subsequent enhancement of total cellular luminescence that eventually mimicked PXR-driven luciferase induction. These data show that particular compounds can strikingly interfere with firefly luciferase and once more illustrate the importance of careful interpretation of data obtained from luciferase-based assays. PMID:27325500

  15. [Construction and function identification of luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR].

    PubMed

    Yang, Shuo; Li, Jia-li; Bi, Hui-chang; Zhou, Shou-ning; Liu, Xiao-man; Zeng, Hang; Hu, Bing-fang; Huang, Min

    2016-01-01

    This study aims to investigate the function of two SNPs (rs8904C > T and rs696G >A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3-vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P < 0.001). For rs696G > A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P < 0.05) and 56.1% (P < 0.001) lower than those containing G allele (pGL3-rs8904C/rs696G and pGL3-rs8904T/rs696G), respectively. For rs8904C > T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C >T didn't have much effect on the luciferase activity. PMID:27405166

  16. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    SciTech Connect

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel . E-mail: octave@nchm.ucl.ac.be

    2007-09-21

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.

  17. The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

    PubMed

    Markova, Svetlana V; Larionova, Marina D; Burakova, Ludmila P; Vysotski, Eugene S

    2015-01-30

    Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 SS bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of ∼3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. PMID:25543059

  18. Functional multiplex reporter assay using tagged Gaussia luciferase

    PubMed Central

    van Rijn, Sjoerd; Nilsson, Jonas; Noske, David P.; Vandertop, W. Peter; Tannous, Bakhos A.; Würdinger, Thomas

    2013-01-01

    We have developed a multiplex reporter system to monitor multiple biological variables in real-time. The secreted Gaussia luciferase was fused to ten different epitope tags (Gluctag), each expressed in different tumor cells. By immunobinding of the tags followed by Gluctag detection, this system allowed the independent and real-time monitoring of mixed cell cultures in vitro and of mixed subcutaneous and intracranial tumor subpopulations in vivo. PMID:23308339

  19. Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

    PubMed

    Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

    2016-02-01

    Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways. PMID:26646257

  20. Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3.

    PubMed

    Yasunaga, Mayu; Murotomi, Kazutoshi; Abe, Hiroko; Yamazaki, Tomomi; Nishii, Shigeaki; Ohbayashi, Tetsuya; Oshimura, Mitsuo; Noguchi, Takako; Niwa, Kazuki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

    2015-01-20

    Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2=9.8 to 2.8h), the half-life of C9-fused ELuc was significantly shorter (t1/2=1.0h) than that of PEST-fused ELuc when measurements were conducted at 37°C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence. PMID:25528501

  1. A route from darkness to light: emergence and evolution of luciferase activity in AMP-CoA-ligases inferred from a mealworm luciferase-like enzyme.

    PubMed

    Viviani, V R; Prado, R A; Neves, D R; Kato, D; Barbosa, J A

    2013-06-11

    The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have

  2. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

    PubMed

    Pazzagli, M; Devine, J H; Peterson, D O; Baldwin, T O

    1992-08-01

    The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT. PMID:1443530

  3. Development of HSPA1A promoter-driven luciferase reporter gene assays in human cells for assessing the oxidative damage induced by silver nanoparticles.

    PubMed

    Xin, Lili; Wang, Jianshu; Zhang, Leshuai W; Che, Bizhong; Dong, Guangzhu; Fan, Guoqiang; Cheng, Kaiming

    2016-08-01

    The exponential increase in the total number of engineered nanoparticles in consumer products requires novel tools for rapid and cost-effective toxicology screening. In order to assess the oxidative damage induced by nanoparticles, toxicity test systems based on a human HSPA1A promoter-driven luciferase reporter in HepG2, LO2, A549, and HBE cells were established. After treated with heat shock and a group of silver nanoparticles (AgNPs) with different primary particle sizes, the cell viability, oxidative damage, and luciferase activity were determined. The time-dependent Ag(+) ions release from AgNPs in cell medium was also evaluated. Our results showed that heat shock produced a strong time-dependent induction of relative luciferase activity in the four luciferase reporter cells. Surprisingly, at 4h of recovery, the relative luciferase activity was >98× the control level in HepG2-luciferase cells. Exposure to different sizes of AgNPs resulted in activation of the HSPA1A promoter in a dose-dependent manner, even at low cytotoxic or non-cytotoxic doses. The smaller (5nm) AgNPs were more potent in luciferase induction than the larger (50 and 75nm) AgNPs. These results were generally in accordance with the oxidative damage indicated by malondialdehyde concentration, reactive oxygen species induction and glutathione depletion, and Ag(+) ions release in cell medium. Compared with the other three luciferase reporter cells, the luciferase signal in HepG2-luciferase cells is obviously more sensitive and stable. We conclude that the luciferase reporter cells, especially the HepG2-luciferase cells, could provide a valuable tool for rapid screening of the oxidative damage induced by AgNPs. PMID:27211842

  4. Identification, characterization and use of two tick promoters for construction of a dual luciferase reporter vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dual luciferase reporter systems are valuable tools for functional genomic studies, but have not previously been developed for use in tick cell culture. We evaluated expression of available luciferase constructs in tick cell cultures derived from Rhipicephalus (Boophilus) microplus, an important vec...

  5. A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.

    PubMed

    Xiao, Dan; Zhang, Weifeng; Li, Yan; Liu, Kuan; Zhao, Junli; Sun, Xiaohong; Shan, Linlin; Mao, Qinwen; Xia, Haibin

    2016-02-10

    Sox2 is an important transcriptional factor that has multiple functions in stem cell maintenance and tumorigenesis. To investigate the transcriptional regulation of the Sox2 gene, a luciferase knock-in reporter system was established in HEK293 cells by placing the luciferase gene in the genome under the control of the Sox2 gene promoter using a transcription activator-like effector nuclease (TALEN)-mediated genome editing technique. PCR and Southern blot results confirmed the site-specific integration of a single copy of the exogenous luciferase gene into the genome. To prove the reliability and sensitivity of this novel luciferase knock-in system, a CRISPR/Cas transcription activation system for the Sox2 gene was constructed and applied to the knock-in system. The results indicated that luciferase activity was directly correlated with the activity of the Sox2 endogenous promoter. This novel system will be a useful tool to study the transcriptional regulation of Sox2, and has great potential in medical and industrial applications. PMID:26721181

  6. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.

    PubMed

    Hall, Mary P; Unch, James; Binkowski, Brock F; Valley, Michael P; Butler, Braeden L; Wood, Monika G; Otto, Paul; Zimmerman, Kristopher; Vidugiris, Gediminas; Machleidt, Thomas; Robers, Matthew B; Benink, Hélène A; Eggers, Christopher T; Slater, Michael R; Meisenheimer, Poncho L; Klaubert, Dieter H; Fan, Frank; Encell, Lance P; Wood, Keith V

    2012-11-16

    Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes. PMID:22894855

  7. Monitoring circadian time in rat plasma using a secreted Cypridina luciferase reporter.

    PubMed

    Yamada, Yoshiko; Nishide, Shin-Ya; Nakajima, Yoshihiro; Watanabe, Toshiyuki; Ohmiya, Yoshihiro; Honma, Ken-Ichi; Honma, Sato

    2013-08-15

    A firefly luciferase reporter enabled us to monitor promoter activity in vivo as well as ex vivo; however, this requires a sufficient supply of the substrate luciferin and specific monitoring devices. To overcome these disadvantages, we developed transgenic rats carrying a secreted enzyme Cypridina luciferase (CLuc) reporter under the promoter of clock gene Per2 (Per2-CLuc). Per2-CLuc activity in serially sampled blood from freely moving rats exhibited robust circadian rhythms with a peak at early morning. The Per2-CLuc bioluminescence could be quantified even with approximately 100pl of plasma. Plasma Per2-CLuc rhythms were phase reversed, and the level was reduced by restricting food access for 2h during the light phase, suggesting that the plasma Per2-CLuc rhythms reflect the phase of peripheral clocks entrained to feeding cues as well as fuel metabolism. Fasting for 2days did not alter the circadian Per2-CLuc rhythms in rats, suggesting that feeding per se did not affect the circadian Per2-CLuc rhythms. Tissue-specific Per2-CLuc rhythms were observed in culture medium of peripheral tissues. The Per2-CLuc reporter is a powerful tool to access gene expression in vivo as well as ex vivo with ordinary laboratory equipment. PMID:23624321

  8. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase.

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

    Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. PMID:16185065

  9. Development of stable HSPA1A promoter-driven luciferase reporter HepG2 cells for assessing the toxicity of organic pollutants present in air.

    PubMed

    Xin, Lili; Li, Xiaohai; Deng, Huaxin; Kuang, Dan; Dai, Xiayun; Huang, Suli; Wang, Feng; He, Meian; Currie, R William; Wu, Tangchun

    2012-09-01

    HSPA1A (HSP70-1) is a highly inducible heat shock gene up-regulated in response to environmental stresses and pollutants. The aim of our study was to evaluate the sensitivity of the stable metabolically competent HepG2 cells containing a human HSPA1A promoter-driven luciferase reporter (HepG2-luciferase cells) for assessing the toxicity of organic pollutants present in air. The HepG2-luciferase cells were validated by heat shock treatment and testing three organic compounds (pyrene, benzo[a]pyrene, and formaldehyde) that are ubiquitous in the air. The maximal level of HSPA1A (HSP70-1) and relative luciferase activity induced by heat shock were over three and nine times the control level, respectively. Pyrene, benzo[a]pyrene, and formaldehyde all induced significantly elevated levels of relative luciferase activity in a dose-dependent manner. Extractable organic matter (EOM) from urban traffic and coke oven emissions in ambient air were tested on the HepG2-luciferase cells. The traffic EOM induced significant increase in relative luciferase activity at concentrations of picogram per liter. The coke oven EOM produced a strong dose-dependent induction of relative luciferase activity up to six times the control value. Significant increases in relative luciferase activity were observed at concentrations that were as low, or lower than the concentrations that the tested organic pollutants decreased cell viability, and increased malondialdehyde concentration, Olive tail moment, and micronuclei frequency. Therefore, we conclude that the HepG2-luciferase cells are a valuable tool for rapid screening of the overall toxicity of organic pollutants present in air. PMID:22367790

  10. Dual luciferase assay for secreted luciferases based on Gaussia and NanoLuc.

    PubMed

    Heise, Kerstin; Oppermann, Henry; Meixensberger, Jürgen; Gebhardt, Rolf; Gaunitz, Frank

    2013-05-01

    Just recently, NanoLuc, a new engineered luciferase based on the small subunit of the luciferase from Oplophorus gracilirostris was introduced. Like the luciferase from Gaussia princeps, this luciferase is secreted into the medium. Both luciferases are the smallest and brightest luciferases known and well-suited for reporter assays. In our experiments, we demonstrate that both luciferases can be used together in a dual-reporter assay by solving the problem that NanoLuc produces a significant signal with coelenterazine, which is the substrate for Gaussia luciferase. We found that the background signal from NanoLuc with coelenterazine can be calculated from the determination of NanoLuc activity in the presence of its substrate furimazine. This in turn allows the precise determination of the activity of Gaussia which does not produce light in the presence of furimazine. Based on this observation, we developed a high sensitive dual secreted luciferase assay which allows the determination of both activities in a single cotransfection experiment. We demonstrate the versatility and robustness of the assay for the normalization of reporter gene activities. Since Gaussia luciferase and NanoLuc are nonhomologous reporters, the method to determine both luciferase activities may also be useful for coincidence reporter gene systems for high-throughput screening. PMID:23679848

  11. Click beetle luciferases as dual reporters of gene expression in Candida albicans.

    PubMed

    Kapitan, Mario; Eichhof, Isabel; Lagadec, Quentin; Ernst, Joachim F

    2016-08-01

    Synthetic genes encoding functional luciferases of the click beetle (CB) Pyrophorus plagiophthalamus have been expressed in the human fungal pathogen Candida albicans. Both green- and red-emitting CB luciferases (CaCBGluc and CaCBRluc) were produced with high efficiency in transformants under transcriptional control of the growth-dependent ACT1 promoter, as well as by the HWP1 and UME6 promoters, which are upregulated during hyphal morphogenesis, as well as by the YWP1 and EFG1 promoters, which are downregulated. For all hyphally regulated genes, relative bioluminescence values derived from promoter fusions approximated relative transcript levels of native genes, although downregulation of YWP1 promoter activity required correction for the stability of CB luciferases (approximate half-lives 30 min for CaCBRluc and 80 min for CaCBGluc, as determined by immunoblotting). Importantly, the activity of both luciferases could be separately monitored in a single strain, in intact cells, in lysed cells or in cell extracts using luciferin as single substrate and inhibition of hypha formation by farnesol could be easily detected by the HWP1p-CaCBRluc fusion. The results suggest that CB luciferases are convenient tools to measure gene expression in C. albicans and may facilitate screenings for antifungal compounds. PMID:27339610

  12. Novel screening method for potential skin-whitening compounds by a luciferase reporter assay.

    PubMed

    Shirasugi, Ichiro; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Matsui, Takashi; Liu, Ming-Cheh; Suiko, Masahito

    2010-01-01

    Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds. PMID:21071833

  13. NanoLuc reporter for dual luciferase imaging in living animals.

    PubMed

    Stacer, Amanda C; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E; Rehemtulla, Alnawaz; Luker, Gary D

    2013-10-01

    Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. PMID:24371848

  14. A Novel HIV-1 Reporter Virus with a Membrane-Bound Gaussia princeps Luciferase

    PubMed Central

    Suree, Nuttee; Koizumi, Naoya; Sahakyan, Anna; Shimizu, Saki; An, Dong Sung

    2014-01-01

    Summary HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1NL4-3 genome using a picornaviral 2A-like sequence. The resulting HIV-1NL4-3mGluc virus expresses Gaussia princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity. PMID:22483780

  15. Approaches to engineer stability of beetle luciferases

    PubMed Central

    Koksharov, Mikhail I.; Ugarova, Natalia N.

    2012-01-01

    Luciferase enzymes from fireflies and other beetles have many important applications in molecular biology, biotechnology, analytical chemistry and several other areas. Many novel beetle luciferases with promising properties have been reported in the recent years. However, actual and potential applications of wild-type beetle luciferases are often limited by insufficient stability or decrease in activity of the enzyme at the conditions of a particular assay. Various examples of genetic engineering of the enhanced beetle luciferases have been reported that successfully solve or alleviate many of these limitations. This mini-review summarizes the recent advances in development of mutant luciferases with improved stability and activity characteristics. It discusses the common limitations of wild-type luciferases in different applications and presents the efficient approaches that can be used to address these problems. PMID:24688645

  16. Secreted Gaussia princeps Luciferase as a Reporter of Escherichia coli Replication in a Mouse Tissue Cage Model of Infection

    PubMed Central

    Liu, Mingyu; Blinn, Christina; McLeod, Sarah M.; Wiseman, John W.; Newman, Joseph V.; Fisher, Stewart L.; Walkup, Grant K.

    2014-01-01

    Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection. PMID:24595353

  17. Effects of nanomaterials on luciferase with significant protection and increased enzyme activity observed for zinc oxide nanomaterials.

    PubMed

    Barber, S; Abdelhakiem, M; Ghosh, K; Mitchell, L; Spidle, R; Jacobs, B; Washington, L; Li, J; Wanekaya, A; Glaspell, G; DeLong, R K

    2011-12-01

    This principle goal of this research was to examine the effects of various nanomaterials on the activity and behavior of the firefly enzyme luciferase. Nanomaterials have been found to stabilize, and in some instances, shown to increase the activity of enzymes. In this study gold, manganese oxide (MnO), and zinc oxide (ZnO) nanomaterials were utilized in order to test their effects on enzyme activity. Luciferase was used because its activity is easy to analyze, as it typically produces a large amount of bioluminescence easily detected by a Microtiter plate reader. Following incubation with the various nanomaterials, luciferase was subjected to degradation by several protein denaturing agents, such as heat, SDS, urea, ethanol, protease, hydrogen peroxide, and pH changes. Results indicated that luciferase activity is indeed affected when combined with nanomaterials, accompanied by both increases and decreases in enzyme activity depending on the type of nanomaterial and denaturing agent used. In most of the experiments, when incubated with ZnO nanomaterials, luciferase depicted significant increases in activity and bioluminescence. Additional experiments, in which human A375 cells were treated with luciferase-nanomaterial mixtures, also depicted increased enzyme activity and bioluminescence for luciferase incubated with ZnO nanomaterials. Ultimately, our findings indicated that when luciferase was subjected to multiple types of denaturation, zinc oxide nanomaterials dramatically preserved and increased enzyme activity and bioluminescence. PMID:22408903

  18. Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

    PubMed

    Ideguchi, Yamato; Oshikoshi, Yuta; Ryo, Masashi; Motoki, Shogo; Kuwano, Takashi; Tezuka, Takafumi; Aoki, Setsuyuki

    2016-01-01

    We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells. PMID:26506945

  19. Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

    NASA Astrophysics Data System (ADS)

    Hundt, Walter; Schink, Christian; Steinbach, Silke; O'Connell-Rodwell, Caitlin E.; Kiessling, Andreas; Librizzi, Damiano; Burbelko, Mykhaylo; Guccione, Samira

    2012-06-01

    We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm3. Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8+/-103.4 mm2 at the beginning versus 704.8+/-94.4 mm3 at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9%+/-4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5+/-0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

  20. Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

    PubMed Central

    Mofford, David M.; Reddy, Gadarla Randheer; Miller, Stephen C.

    2014-01-01

    Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation. PMID:24616520

  1. Interaction of firefly luciferase and silver nanoparticles and its impact on enzyme activity

    NASA Astrophysics Data System (ADS)

    Käkinen, Aleksandr; Ding, Feng; Chen, Pengyu; Mortimer, Monika; Kahru, Anne; Ke, Pu Chun

    2013-08-01

    We report on the dose-dependent inhibition of firefly luciferase activity induced by exposure of the enzyme to 20 nm citrate-coated silver nanoparticles (AgNPs). The inhibition mechanism was examined by characterizing the physicochemical properties and biophysical interactions of the enzyme and the AgNPs. Consistently, binding of the enzyme induced an increase in zeta potential from -22 to 6 mV for the AgNPs, triggered a red-shift of 44 nm in the absorbance peak of the AgNPs, and rendered a ‘protein corona’ of 20 nm in thickness on the nanoparticle surfaces. However, the secondary structures of the enzyme were only marginally affected upon formation of the protein corona, as verified by circular dichroism spectroscopy measurement and multiscale discrete molecular dynamics simulations. Rather, inductively coupled plasma mass spectrometry measurement revealed a significant ion release from the AgNPs. The released silver ions could readily react with the cysteine residues and N-groups of the enzyme to alter the physicochemical environment of their neighboring catalytic site and subsequently impair the enzymatic activity.

  2. Application of Gaussia luciferase in bicistronic and non-conventional secretion reporter constructs

    PubMed Central

    2014-01-01

    Background Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail. Results We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC. Conclusions Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system. PMID:25007711

  3. Simultaneous analysis of the bidirectional African cassava mosaic virus promoter activity using two different luciferase genes.

    PubMed

    Frey, P M; Schärer-Hernández, N G; Fütterer, J; Potrykus, I; Puonti-Kaerlas, J

    2001-03-01

    The expression of geminivirus genes is controlled by bidirectional promoters which are located in the large intergenic region of the circular DNA genomes and specifically regulated by virus encoded proteins. In order to study the simultaneous regulation of both orientations of the DNA A and DNA B promoters of African cassava mosaic virus (ACMV), they were cloned between two different luciferase genes with the firefly luciferase gene in complementary-sense and the Renilla luciferase gene in virion-sense orientation. The regulation of the ACMV promoters by proteins encoded by the complete DNA A, as well as by the individually expressed transactivator (TrAP) or replication-associated (Rep) proteins was assessed in tobacco and cassava protoplasts using dual luciferase assays. In addition, the regulation of the DNA A promoter integrated into tobacco genome was also assessed. The results show that TrAP activates virion-sense expression strongly both in cassava and tobacco protoplasts, but not in transgenic tobacco plants. In contrast to this, DNA A encoded proteins activate virion-sense expression both in protoplasts and in transgenic plants. At the same time they reduce the expression of the complementary-sense Rep gene on DNA A but activate the expression of the complementary-sense movement protein (MPB) gene on DNA B. The degree of MBP activation is higher in cassava than in tobacco protoplasts, indicating that the plant host also influences the promoter strength. Transient transformation experiments using linearized DNA indicate that the different regulation of the ACMV DNA A promoter in protoplasts and transgenic plants could be due to different DNA curvature in free plasmids and in genes integrated in plant genomic DNA. PMID:11324760

  4. Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity

    PubMed Central

    Endo, Takeshi; Noda, Natsumi; Kuromi, Yasushi; Kokura, Kenji; Kazuki, Yasuhiro; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2016-01-01

    Background Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. Methods We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. Results The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. Conclusion By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions. PMID:27493490

  5. Analysis of structural changes in active site of luciferase adsorbed on nanofabricated hydrophilic Si surface by molecular-dynamics simulations

    SciTech Connect

    Nishiyama, Katsuhiko; Hoshino, Tadatsugu

    2007-05-21

    Interactions between luciferase and a nanofabricated hydrophilic Si surface were explored by molecular-dynamics simulations. The structural changes in the active-site residues, the residues affecting the luciferin binding, and the residues affecting the bioluminescence color were smaller on the nanofabricated hydrophilic Si surface than on both a hydrophobic Si surface and a hydrophilic Si surface. The nanofabrication and wet-treatment techniques are expected to prevent the decrease in activity of luciferase on the Si surface.

  6. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

  7. Generation of recombinant rabies viruses encoding NanoLuc luciferase for antiviral activity assays.

    PubMed

    Anindita, Paulina Duhita; Sasaki, Michihito; Nobori, Haruaki; Sato, Akihiko; Carr, Michael; Ito, Naoto; Sugiyama, Makoto; Orba, Yasuko; Sawa, Hirofumi

    2016-04-01

    Rabies is an invariably fatal disease caused by Rabies virus (RABV), a member of the family Rhabdoviridae, genus Lyssavirus. Once central nervous infection occurs and symptoms develop, the case fatality rate approaches 100% despite availability of post-exposure prophylaxis. Therefore, new antiviral therapies for rabies are urgently required. Antivirals which can inhibit virus replication can be identified through screening of small compounds, however, as RABV infection does not generate easily discernible cytopathic effects in vitro, cell viability assays may not be feasible to observe antiviral activity of small compounds against RABV. In this study, recombinant RABVs (rRABVs) encoding NanoLuc luciferase (NanoLuc) were generated to facilitate the screening of small compound libraries. NanoLuc expression was confirmed in single-step growth cures of virus infection and showed that the rRABVs were capable of viral replication without decrease of luciferase activity through ten serial passages. Furthermore, the rRABVs were able to quantify the antiviral activity of the nucleoside analogue ribavirin against RABV in vitro. These findings confirm the potential of the rRABV encoding NanoLuc system to facilitate screening of small compounds to inhibit RABV infection. PMID:26869397

  8. Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae.

    PubMed

    Masser, Anna E; Kandasamy, Ganapathi; Kaimal, Jayasankar Mohanakrishnan; Andréasson, Claes

    2016-05-01

    Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo® is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE ), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate. © 2016 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:26860732

  9. A highly sensitive assay of IRE1 activity using the small luciferase NanoLuc: Evaluation of ALS-related genetic and pathological factors.

    PubMed

    Hikiji, Takahiro; Norisada, Junpei; Hirata, Yoko; Okuda, Kensuke; Nagasawa, Hideko; Ishigaki, Shinsuke; Sobue, Gen; Kiuchi, Kazutoshi; Oh-hashi, Kentaro

    2015-08-01

    Activation of inositol-requiring enzyme 1 (IRE1) due to abnormal conditions of the endoplasmic reticulum (ER) is responsible for the cleavage of an unspliced form of X-box binding protein 1 (uXBP1), producing its spliced form (sXBP1). To estimate IRE1 activation, several analytical procedures using green fluorescence protein and firefly luciferase have been developed and applied to clarify the roles of IRE1-XBP1 signaling pathways during development and disease progression. In this study, we established a highly sensitive assay of IRE1 activity using a small luciferase, NanoLuc, which has approximately 100-fold higher activity than firefly luciferase. The NanoLuc reporter, which contained a portion of the spliced region of XBP1 upstream of NanoLuc, was highly sensitive and compatible with several types of cell lines. We found that NanoLuc was secreted into the extracellular space independent of the ER-Golgi pathway. The NanoLuc activity of an aliquot of culture medium from the neuroblastoma-spinal neuron hybrid cell line NSC-34 reflected the toxic stimuli-induced elevation of intracellular activity well. Using this technique, we evaluated the effects of several genetic and pathological factors associated with the onset and progression of amyotrophic lateral sclerosis (ALS) on NanoLuc reporter activity. Under our experimental conditions, inhibition of ER-Golgi transport by the overexpression of mutant Sar1 activated luciferase activity, whereas the co-expression of mutant SOD1 or the C-terminal fragment of TDP-43 (TDP-25) did not. The addition of homocysteine elevated the reporter activity; however, we did not observe any synergistic effect due to the overexpression of the mutant genes described above. Taken together, these data show that our analytical procedure is highly sensitive and convenient for screening useful compounds that modulate IRE1-XBP1 signaling pathways as well as for estimating IRE1 activation in several pathophysiological diseases. PMID:26056941

  10. Photographic and luminometric detection of luciferase reporter phages for drug susceptibility testing of clinical Mycobacterium tuberculosis isolates.

    PubMed

    Hazbón, Manzour Hernando; Guarín, Nora; Ferro, Beatriz Eugenia; Rodríguez, Ana Lucía; Labrada, Luz Angela; Tovar, Rafael; Riska, Paul F; Jacobs, William R

    2003-10-01

    Luciferase reporter phages (LRPs) have proven to be efficient tools for drug susceptibility testing of Mycobacterium tuberculosis. Luminometric detection of LRP activity offers higher sensitivity and quantitative results, while a Polaroid film detection method offers a "low-tech" inexpensive alternative that is called the Bronx box. In this work we evaluated, improved, and compared the performance of the luminometer and the Bronx box formats for drug susceptibility testing with LRPs by using 51 clinical isolates of M. tuberculosis, with the agar proportion method (PM) serving as reference. The sensitivity in detecting resistance to isoniazid and rifampin, antibiotics that define multidrug resistance (MDR), was 100% for both methods. The turnaround time for results was reduced from 3 weeks for PM to 54 or 94 h for luminometry or the Bronx box, respectively. These results support the utility of LRPs as a screening test for the surveillance of MDR tuberculosis. PMID:14532245

  11. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases. PMID:21505686

  12. Bioorthogonal Catalysis: A General Method To Evaluate Metal-Catalyzed Reactions in Real Time in Living Systems Using a Cellular Luciferase Reporter System.

    PubMed

    Hsu, Hsiao-Tieh; Trantow, Brian M; Waymouth, Robert M; Wender, Paul A

    2016-02-17

    The development of abiological catalysts that can function in biological systems is an emerging subject of importance with significant ramifications in synthetic chemistry and the life sciences. Herein we report a biocompatible ruthenium complex [Cp(MQA)Ru(C3H5)](+)PF6(-) 2 (Cp = cyclopentadienyl, MQA = 4-methoxyquinoline-2-carboxylate) and a general analytical method for evaluating its performance in real time based on a luciferase reporter system amenable to high throughput screening in cells and by extension to evaluation in luciferase transgenic animals. Precatalyst 2 activates alloc-protected aminoluciferin 4b, a bioluminescence pro-probe, and releases the active luminophore, aminoluciferin (4a), in the presence of luciferase-transfected cells. The formation and enzymatic turnover of 4a, an overall process selected because it emulates pro-drug activation and drug turnover by an intracellular target, is evaluated in real time by photon counting as 4a is converted by intracellular luciferase to oxyaminoluciferin and light. Interestingly, while the catalytic conversion (activation) of 4b to 4a in water produces multiple products, the presence of biological nucleophiles such as thiols prevents byproduct formation and provides almost exclusively luminophore 4a. Our studies show that precatalyst 2 activates 4b extracellularly, exhibits low toxicity at concentrations relevant to catalysis, and is comparably effective in two different cell lines. This proof of concept study shows that precatalyst 2 is a promising lead for bioorthogonal catalytic activation of pro-probes and, by analogy, similarly activatable pro-drugs. More generally, this study provides an analytical method to measure abiological catalytic activation of pro-probes and, by analogy with our earlier studies on pro-Taxol, similarly activatable pro-drugs in real time using a coupled biological catalyst that mediates a bioluminescent readout, providing tools for the study of imaging signal amplification

  13. Structure–function studies on the active site of the coelenterazine-dependent luciferase from Renilla

    PubMed Central

    Woo, Jongchan; Howell, Matthew H.; von Arnim, Albrecht G.

    2008-01-01

    Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the α/β hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220—three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB—also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate. PMID:18359861

  14. Illuminating Cancer Systems With Genetically-Engineered Mouse Models and Coupled Luciferase Reporters In Vivo

    PubMed Central

    Kocher, Brandon; Piwnica-Worms, David

    2013-01-01

    Bioluminescent imaging (BLI) is a powerful non-invasive tool that has dramatically accelerated the in vivo interrogation of cancer systems and longitudinal analysis of mouse models of cancer over the past decade. Various luciferase enzymes have been genetically engineered into mouse models (GEMMs) of cancer which permit investigation of cellular and molecular events associated with oncogenic transcription, post-transcriptional processing, protein-protein interactions, transformation and oncogene addiction in live cells and animals. Luciferase-coupled GEMMs ultimately serve as a non-invasive, repetitive, longitudinal, and physiological means by which cancer systems and therapeutic responses can be investigated accurately within the autochthonous context of a living animal. PMID:23585416

  15. A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections.

    PubMed

    Bacconi, Marta; Haag, Andreas F; Torre, Antonina; Castagnetti, Andrea; Chiarot, Emiliano; Delany, Isabel; Bensi, Giuliano

    2016-04-01

    In vivo imaging of bioluminescent bacteria permits their visualization in infected mice, allowing spatial and temporal evaluation of infection progression. Most available bioluminescent strains were obtained by integration of the luciferase genes into the bacterial chromosome, a challenging and time-consuming approach. Recently, episomal plasmids were used, which were introduced in bacteria and expressed all genes required for bioluminescence emission. However, the plasmid was progressively lost in vitro and in vivo, if bacteria were not maintained under antibiotic selective pressure. Increased stability could be obtained inserting into the plasmid backbone sequences that assured plasmid partition between daughter bacterial cells, or caused death of bacteria that had lost the plasmid. So far, no detailed analysis was performed of either plasmid stability in vivo or contribution of different stabilizing sequence types. Here we report the construction of a plasmid, which includes the Photorhabdus luminescens lux cassette expressed under the control of a Staphylococcus aureus specific gene promoter, and toxin/antitoxin (T/A) and partition sequences (Par) conferring stability and transmissibility of the plasmid. Following infection of mice with S. aureus carrying this plasmid, we demonstrated that the promoter-lux fusion was functional in vivo, that the plasmid was retained by 70-100% of bacterial cells 7 days post-infection, and that both stabilizing sequence types were required to maximize plasmid retention. These data suggest that the plasmid can be a valuable tool to study gene expression and bacterial spread in small laboratory animals infected with S. aureus or possibly other Gram-positive human pathogens. PMID:26685857

  16. Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

    PubMed

    Alberti, Michael O; Jones, Jennifer J; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K; Edmonds, Tara G; Kappes, John C; Ochsenbauer, Christina

    2015-12-01

    We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus

  17. Transgenic Mouse Model Harboring the Transcriptional Fusion Ccl20-Luciferase as a Novel Reporter of Pro-Inflammatory Response

    PubMed Central

    Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

    2013-01-01

    The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo. PMID:24265691

  18. Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response.

    PubMed

    Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

    2013-01-01

    The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo. PMID:24265691

  19. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    2008-08-29

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

  20. Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

    PubMed Central

    Stefan, E.; Aquin, S.; Berger, N.; Landry, C. R.; Nyfeler, B.; Bouvier, M.; Michnick, S. W.

    2007-01-01

    The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Gαs protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive β-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades. PMID:17942691

  1. The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

    PubMed

    Prado, R A; Santos, C R; Kato, D I; Murakami, M T; Viviani, V R

    2016-05-11

    Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343). PMID:27101527

  2. Imaging Dendrimer-Grafted Graphene Oxide Mediated Anti-miR-21 Delivery With an Activatable Luciferase Reporter.

    PubMed

    Wang, Fu; Zhang, Beilei; Zhou, Lin; Shi, Yaru; Li, Zhiqiang; Xia, Yuqiong; Tian, Jie

    2016-04-13

    MicroRNAs (miRNAs) are a class of post-transcriptional gene regulators involved in various physiological processes including carcinogenesis, and they have emerged as potential targets for tumor theranostics. However, the employment of antisense oligonucleotides, termed anti-miRs, for antagonizing miRNA functions in vivo has largely been impeded by a lack of effective delivery carriers. Here, we describe the development of polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG)-functionalized nanographene oxide (NGO) conjugate (NGO-PEG-dendrimer) for the efficient delivery of anti-miR-21 into non-small-cell lung cancer cells. To monitor the delivery of anti-miR-21 into cells and tumors, we also constructed an activatable luciferase reporter (Fluc-3xPS) containing three perfectly complementary sequences against miR-21 in the 3' untranslated region (UTR) of the reporter. Compared with bare dendrimer and Lipofectamine 2000 (Lipo2000), NGO-PEG-dendrimer showed considerably lower cytotoxicity and higher transfection efficiency. As demonstrated by in vitro bioluminescence imaging and Western blotting assays, NGO-PEG-dendrimer effectively delivered anti-miR-21 into the cytoplasm and resulted in the upregulation of luciferase intensity and PTEN target protein expression in a dose-dependent manner. Moreover, transfection with anti-miR-21 by NGO-PEG-dendrimer led to stronger inhibition of cell migration and invasion than did bare dendrimer or Lipo2000 transfection. The intravenous delivery of anti-miR-21 via NGO-PEG-dendrimer induced a significant increase in the bioluminescence signal within the Fluc-3xPS reporter-transplanted tumor areas. These results suggest that NGO-PEG-dendrimer could be an efficient and a potential nanocarrier for delivering RNA oligonucleotides. In addition, the strategy of combining NGO-PEG-dendrimer with an activatable luciferase reporter allows the image-guided monitoring of the delivery process, which can provide insights into the RNA

  3. Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

    NASA Astrophysics Data System (ADS)

    Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J. Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

    2016-02-01

    Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies.Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery

  4. Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter.

    PubMed

    Wang, Fu; Yang, Kai; Wang, Zhe; Ma, Ying; Gutkind, J Silvio; Hida, Naoki; Niu, Gang; Tian, Jie

    2016-02-21

    Imaging guided techniques have been increasingly employed to investigate the pharmacokinetics (PK) and biodistribution of nanoparticle based drug delivery systems. In most cases, however, the PK profiles of drugs could vary significantly from those of drug delivery carriers upon administration in the blood circulation, which complicates the interpretation of image findings. Herein we applied a genetically encoded luciferase reporter in conjunction with near infrared (NIR) fluorophores to investigate the respective PK profiles of a drug and its carrier in a biodegradable drug delivery system. In this system, a prototype hydrophobic agent, rapamycin (Rapa), was encapsulated into human serum albumin (HSA) to form HSA Rapa nanoparticles, which were then labeled with Cy5 fluorophore to facilitate the fluorescence imaging of HSA carrier. Meanwhile, we employed transgenetic HN12 cells that were modified with a split luciferase reporter, whose bioluminescence function is regulated by Rapa, to reflect the PK profile of the encapsulated agent. It was interesting to discover that there existed an obvious inconsistency of PK behaviors between HSA carrier and rapamycin in vitro and in vivo through near infrared fluorescence imaging (NIFRI) and bioluminescence imaging (BLI) after treatment with Cy5 labeled HSA Rapa. Nevertheless, HSA Rapa nanoparticles manifested favorable in vivo PK and tumor suppression efficacy in a follow-up therapeutic study. The developed strategy of combining a molecular reporter and a fluorophore in this study could be extended to other drug delivery systems to provide profound insights for non-invasive real-time evaluation of PK profiles of drug-loaded nanoparticles in pre-clinical studies. PMID:26818100

  5. Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box.

    PubMed

    Riska, P F; Su, Y; Bardarov, S; Freundlich, L; Sarkis, G; Hatfull, G; Carrière, C; Kumar, V; Chan, J; Jacobs, W R

    1999-04-01

    Detecting antibiotic resistance in Mycobacterium tuberculosis is becoming increasingly important with the global recognition of drug-resistant strains and their adverse impact on clinical outcomes. Current methods of susceptibility testing are either time-consuming or costly; rapid, reliable, simple, and inexpensive methods would be highly desirable, especially in the developing world where most tuberculosis is found. The luciferase reporter phage is a unique reagent well-suited for this purpose: upon infection with viable mycobacteria, it produces quantifiable light which is not observed in mycobacterial cells treated with active antimicrobials. In this report, we describe a modification of our original assay, which allows detection of the emitted light with a Polaroid film box designated the Bronx Box. The technique has been applied to 25 M. tuberculosis reference and clinical strains, and criteria are presented which allow rapid and simple discrimination among strains susceptible or resistant to isoniazid and rifampin, the major antituberculosis agents. PMID:10074539

  6. A luciferase reporter gene assay and aryl hydrocarbon receptor 1 genotype predict the LD{sub 50} of polychlorinated biphenyls in avian species

    SciTech Connect

    Manning, Gillian E.; Farmahin, Reza; Crump, Doug; Jones, Stephanie P.; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

    2012-09-15

    Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R{sup 2} ≥ 0.87, p < 0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals. -- Highlights: ► PCB embryolethality in birds can be predicted from a species' AHR1 genotype. ► The reporter gene assay is useful for predicting species sensitivity to PCBs. ► The relative potency of PCBs does not appear to differ between AHR1 genotypes. ► Contamination of PCB 105 and PCB 118 did not affect their

  7. [Subunit interactions in luciferase from the firefly Luciola mingrelica. Their role in the manifestation of enzyme activity and during thermoinactivation].

    PubMed

    Brovko, L Iu; Beliaeva, E I; Ugarova, N N

    1982-05-01

    It was shown that the dimers of the firefly luciferase possess the catalytic activity, whereas the monomers do not. The dissociation constant (Kd) for active dimers was determined at pH 7.0--8.4 within the temperature range of 15--35 degrees and at MgSO4 and Na2SO4 concentrations varying from 37 to 370 mM and 49 to 490 mM, respectively. Under variable conditions the Kd value changed only insignificantly and made up to 13 nm. The substitution of Na2SO4 for MgSO4 decreased Kd 2.5 times. The effective rate constant for the enzyme inactivation (kin) was increased more than 5-fold, when the luciferase concentration was decreased from 200 down to 3.5 nM in the presence of 37 mM MgSO4. When the concentration of the latter was increased up to 185 mM, the value of kin ceased to depend on the enzyme concentration. The decrease of kin was also observed at an increase in Na2SO4. An inactivation pattern for the enzyme in solution was determined both for the monomer and for the dimer of the enzyme. The equations allowing to calculate the inactivation constant for the monomer (Ki) and dimer (k2) at different pH values, temperatures and salt concentrations were obtained. The enzyme was found to be stabilized by salts more than 10-fold, the stabilizing effect being far more pronounced for the enzyme monomer than for the dimer. The dependence of the effective kin value on pH and temperature was primarily influenced by the dependence of the inactivation rate constant for the dimer. PMID:7093378

  8. Supramolecular Control over Split-Luciferase Complementation.

    PubMed

    Bosmans, Ralph P G; Briels, Jeroen M; Milroy, Lech-Gustav; de Greef, Tom F A; Merkx, Maarten; Brunsveld, Luc

    2016-07-25

    Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks. PMID:27356091

  9. Comparison of firefly luciferase and NanoLuc luciferase for biophotonic labeling of group A Streptococcus.

    PubMed

    Loh, Jacelyn M S; Proft, Thomas

    2014-04-01

    NanoLuc luciferase (Nluc) is an engineered enzyme that catalyses the substrate, furimazine, to produce light. Nluc has higher sensitivity than the commonly used bioluminescent reporter, firefly luciferase (FFluc). We have introduced Nluc into a toxin-antitoxin stabilised plasmid for the efficient labeling of group A Streptococcus. Comparison of signal strength and kinetic properties between Nluc-labeled bacteria and similarly previously-labeled FFluc bacteria, showed that the bioluminescent signal produced by Nluc-labeled bacteria is up to 15-times higher than FFluc-labeled bacteria during the logarithmic phase. However, with Nluc we were unable to differentiate between bacteria that are metabolically active and inactive because of its ATP-independence. Nluc therefore offers a more sensitive reporter but, perhaps, one more restricted for downstream applications. PMID:24322775

  10. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

    PubMed Central

    Wigdal, Susan S; Anderson, Jessica L; Vidugiris, Gediminas J; Shultz, John; Wood, Keith V; Fan, Frank

    2008-01-01

    Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening. PMID:20161840

  11. Staphylococcus aureus Cell Extract Transcription-Translation Assay: Firefly Luciferase Reporter System for Evaluating Protein Translation Inhibitors

    PubMed Central

    Murray, Robert W.; Melchior, Earline P.; Hagadorn, Jeanne C.; Marotti, Keith R.

    2001-01-01

    The promoter for the Staphylococcus aureus capsule polysaccharide synthesis gene (cap1A) was cloned in front of the firefly luciferase gene for use in a cell extract S. aureus transcription-translation system. The assay is rapid, reproducible, and sensitive and has a lower background level than the radiolabeled amino acid incorporation translation assays. We present data evaluating a transcription inhibitor and a number of protein translation inhibitors in this system. PMID:11353649

  12. Liganded Thyroid Hormone Receptor Inhibits Phorbol 12-O-Tetradecanoate-13-Acetate-Induced Enhancer Activity via Firefly Luciferase cDNA

    PubMed Central

    Misawa, Hiroko; Sasaki, Shigekazu; Matsushita, Akio; Ohba, Kenji; Iwaki, Hiroyuki; Matsunaga, Hideyuki; Suzuki, Shingo; Ishizuka, Keiko; Oki, Yutaka; Nakamura, Hirotoshi

    2012-01-01

    Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity

  13. Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells

    NASA Astrophysics Data System (ADS)

    Liang, Yajie; Walczak, Piotr; Bulte, Jeff W. M.

    2012-01-01

    One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below 600 nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase (Ppy RE9, PRE9) against the yellow luciferase luc2 gene for use in cell transplantation studies. C17.2 neural stem cells expressing PRE9-Venus and luc2-Venus were sorted by flow cytometry and assessed for bioluminescence in vitro in culture and in vivo after transplantation into the brain of immunodeficient Rag2-/- mice. We found that the luminescence from PRE9 was stable, with a peak emission at 620 nm, shifted to the red compared to that of luc2. The emission peak for PRE9 was pH-independent, in contrast to luc2, and much less affected by tissue absorbance compared to that of luc2. However, the total emitted light radiance from PRE9 was substantially lower than that of luc2, both in vitro and in vivo. We conclude that PRE9 has favorable properties as compared to luc2 in terms of pH independence, red-shifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity.

  14. Ah-receptor controlled luciferase expression: A novel species-specific bioassay for Ah-receptor active compounds in environmental matrices

    SciTech Connect

    Murk, A.J.; Aarts, J.M.M.J.G.; Koeman, J.H.; Brouwer, A.; Denison, M.S.

    1995-12-31

    Polyhalogenated aromatic hydrocarbons (PHAHs) such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) are persistent lipophilic compounds that accumulate especially in sediments and in top predators of the aquatic foodchain. PHAHs elicit a number of common toxic responses, which are highly species-specific. The most toxic, planar, PHAHs share a common mechanism of action mediated by the aryl hydrocarbon receptor (AhR). Based on this mechanism, the toxic equivalency factor (TEF) concept has been developed, allowing hazard and risk assessment for mixtures of PHAHs. The TEF-approach assumes additive responses, but also synergistic and antagonistic interactions have been observed. In addition, the often large number of compounds in a mixture, low levels of individual congeners, possible presence of unknown AhR-active substances, and species differences in inducibility, ask for an comprehensive approach in hazard assessment. A number of recombinant cell lines, including Hepa1c1c7 mouse and H411E rat hepatoma cell lines, were developed, showing AhR-mediated firefly (Photinuspyralis) luciferase gene expression. The response by 2,3,7,8-TCDD in the CALUX (chemical activated luciferase expression) assay with these cell lines is dose-dependent, and not subjected to substrate inhibition at higher ligand concentrations. The detection limit for 2,3,7,8-TCDD is below 1 pM (0.2 fmol). The luciferase assay has been successfully applied for monitoring the amount of AhR-active compounds in small aliquots of blood plasma and in both sediment and pore-water samples, of which examples will be presented.

  15. Firefly luciferase gene: structure and expression in mammalian cells.

    PubMed Central

    de Wet, J R; Wood, K V; DeLuca, M; Helinski, D R; Subramani, S

    1987-01-01

    The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression. Images PMID:3821727

  16. Superluminescent variants of marine luciferases for bioassays.

    PubMed

    Kim, Sung Bae; Suzuki, Hideyuki; Sato, Moritoshi; Tao, Hiroaki

    2011-11-15

    In this study, a rational synthesis of superluminescent variants from marine luciferases with prolonged bioluminescence has been demonstrated. A putative active site of a model marine luciferase, Gaussia princeps Luciferase (GLuc), was assigned and modified by a site-directed mutagenesis. The potent variants were found to generate up to 10 times stronger bioluminescence, emitting red shifts of up to 33 nm with natural coelenterazine than native GLuc, rendering an efficient optical signature in bioassays. The advantageous properties were demonstrated with mammalian two-hybrid assays, single-chain probes, and metastases of murine B16 melanoma in BALB/c nude mice. The unique ideas for engineering GLuc are proved to be valid even for other marine luciferases. PMID:21951281

  17. Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors

    PubMed Central

    Samsonov, Andrey; Zenser, Nathan; Zhang, Fan; Zhang, Hongyi; Fetter, John; Malkov, Dmitry

    2013-01-01

    Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN) - mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer

  18. Detection of traces of tetracyclines from fish with a bioluminescent sensor strain incorporating bacterial luciferase reporter genes.

    PubMed

    Pellinen, Teijo; Bylund, Göran; Virta, Marko; Niemi, Anneli; Karp, Matti

    2002-08-14

    Bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline family of antimicrobial agents was optimized to work with fish samples. The biosensing strain contains a plasmid incorporating the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive element from transposon Tn10 (Korpela et al. Anal. Chem. 1998, 70, 4457-4462). The extraction procedure of oxytetracycline from rainbow trout (Oncorhynchus mykiss) tissue was optimized. There was neither need for centrifugation of homogenized tissue nor use of organic solvents. The lowest levels of detection of tetracycline and oxytetracycline from spiked fish tissue were 20 and 50 microg/kg, respectively, in a 2-h assay. The optimized assay protocol was tested with fish that were given a single oral dose of high and low concentrations of oxytetracycline. The assay was able to detect oxytetracycline residues below the European Union maximum residue limits, and the results correlated well with those obtained by conventional HPLC (R = 0.81). PMID:12166964

  19. Hydrophobin-1 promotes thermostability of firefly luciferase.

    PubMed

    Lohrasbi-Nejad, Azadeh; Torkzadeh-Mahani, Masoud; Hosseinkhani, Saman

    2016-07-01

    The thermal sensitivity of firefly luciferase limits its use in certain applications. Firefly luciferase has hydrophobic sites on its surface, which lead to aggregation and inactivation of the enzyme at temperatures over 30 °C. We have successfully stabilized firefly luciferase at high temperatures with the assistance of a unique protein, hydrophobin-1 (HFB1). HFB1 is a small secretory protein belonging to class II of hydrophobins with a low molecular weight (7.5 kDa) and distinct functional hydrophobic patch on its surface. The interaction of HFB1 with hydrophobic sites on the surface of luciferase was confirmed by extrinsic fluorescence studies using 8-anilino-1-naphthalenesulfonic acid (ANS) as a hydrophobic reporter probe. Calculation of thermodynamic parameters of heat inactivation of luciferase shows that conformational changes and flexibility of enzyme decreased in the presence of HFB1, and thermostability of the HFB1-treated enzyme increased. Furthermore, the addition of HFB1 into the enzymatic solution leads to an increase in catalytic efficiency of luciferase and subsequently improves the utility of the enzyme as an ATP detector. PMID:27191938

  20. Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens.

    PubMed

    Yan, Dan; Weisshaar, Marco; Lamb, Kristen; Chung, Hokyung K; Lin, Michael Z; Plemper, Richard K

    2015-09-15

    Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted co-infection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase. PMID:26307636

  1. A novel circadian phenotype based on firefly luciferase expression in transgenic plants.

    PubMed Central

    Millar, A J; Short, S R; Chua, N H; Kay, S A

    1992-01-01

    A 320-bp fragment of the Arabidopsis cab2 promoter is sufficient to mediate transcriptional regulation by both phytochrome and the circadian clock. We fused this promoter fragment to the firefly luciferase (Luc) gene to create a real-time reporter for regulated gene expression in intact plants. Cab2::Luc transcript accumulated in the expected patterns and luciferase activity was closely correlated to cab2::Luc mRNA abundance in both etiolated and green seedlings. The concentration of the bulk of luciferase protein did not reflect these patterns but maintained a relatively constant level, implying that a post-translational mechanism(s) leads to the high-amplitude regulation of luciferase activity. We used a low-light video imaging system to establish that luciferase bioluminescence in vivo accurately reports the temporal and spatial regulation of cab2 transcription in single seedlings. The unique qualities of the firefly luciferase system allowed us to monitor regulated gene expression in real time in individual multicellular organisms. This noninvasive marker for temporal regulation at the molecular level constitutes a circadian phenotype, which may be used to isolate mutants in the circadian clock. PMID:1392609

  2. Coelenterazine-dependent luciferases.

    PubMed

    Markova, S V; Vysotski, E S

    2015-06-01

    Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature - the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo. PMID:26531017

  3. Comparative theoretical study of the binding of luciferyl-adenylate and dehydroluciferyl-adenylate to firefly luciferase

    NASA Astrophysics Data System (ADS)

    Pinto da Silva, Luís; Vieira, João; Esteves da Silva, Joaquim C. G.

    2012-08-01

    This is the first report of a study employing a computational approach to study the binding of (D/L)-luciferyl-adenlyates and dehydroluciferyl-adenylate to firefly luciferase. A semi-empirical/molecular mechanics methodology was used to study the interaction between these ligands and active site molecules. All adenylates are complexed with the enzyme, mostly due to electrostatic interactions with cationic residues. Dehydroluciferyl-adenylate is expected to be a competitive inhibitor of luciferyl-adenylate, as their binding mechanism and affinity to luciferase are very similar. Both luciferyl-adenylates adopt the L-orientation in the active site of luciferase.

  4. Expression and stabilization of bacterial luciferase in mammalian cells

    NASA Astrophysics Data System (ADS)

    Patterson, Stacey S.; Dionisi, Hebe M.; Gupta, Rakesh K.; Sayler, Gary S.

    2004-06-01

    Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

  5. Luciferase expression and bioluminescence does not affect tumor cell growth in vitro or in vivo.

    PubMed

    Tiffen, Jessamy C; Bailey, Charles G; Ng, Cynthia; Rasko, John E J; Holst, Jeff

    2010-01-01

    Live animal imaging is becoming an increasingly common technique for accurate and quantitative assessment of tumor burden over time. Bioluminescence imaging systems rely on a bioluminescent signal from tumor cells, typically generated from expression of the firefly luciferase gene. However, previous reports have suggested that either a high level of luciferase or the resultant light reaction produced upon addition of D-luciferin substrate can have a negative influence on tumor cell growth. To address this issue, we designed an expression vector that allows simultaneous fluorescence and luminescence imaging. Using fluorescence activated cell sorting (FACS), we generated clonal cell populations from a human breast cancer (MCF-7) and a mouse melanoma (B16-F10) cell line that stably expressed different levels of luciferase. We then compared the growth capabilities of these clones in vitro by MTT proliferation assay and in vivo by bioluminescence imaging of tumor growth in live mice. Surprisingly, we found that neither the amount of luciferase nor biophotonic activity was sufficient to inhibit tumor cell growth, in vitro or in vivo. These results suggest that luciferase toxicity is not a necessary consideration when designing bioluminescence experiments, and therefore our approach can be used to rapidly generate high levels of luciferase expression for sensitive imaging experiments. PMID:21092230

  6. High-throughput functional screening using a homemade dual-glow luciferase assay.

    PubMed

    Baker, Jessica M; Boyce, Frederick M

    2014-01-01

    We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

  7. High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

    PubMed Central

    Baker, Jessica M.; Boyce, Frederick M.

    2014-01-01

    We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

  8. Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

    PubMed

    Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

    2016-08-30

    Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

  9. Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli.

    PubMed Central

    González-Flecha, B; Demple, B

    1994-01-01

    Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a

  10. A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

    PubMed

    Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

    2016-05-01

    Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. PMID:26898957

  11. Activity report

    SciTech Connect

    Yu, S W

    2008-08-11

    This report is aimed to show the author's activities to support the LDRD. The title is 'Investigation of the Double-C Behavior in the Pu-Ga Time-Temperature-Transformation Diagram' The sections are: (1) Sample Holder Test; (2) Calculation of x-ray diffraction patterns; (3) Literature search and preparing publications; (4) Tasks Required for APS Experiments; and (5) Communications.

  12. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  13. Luciferase-based protein-denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: developing an intracellular thermometer

    PubMed Central

    Raoof, Mustafa; Zhu, Cihui; Kaluarachchi, Warna D.; Curley, Steven A.

    2013-01-01

    Background Several studies have reported targeted hyperthermia at the cellular level using remote activation of nanoparticles by radiofrequency waves. To date, methods to quantify intracellular thermal dose have not been reported. In this report we study the relationship between radio wave exposure and luciferase denaturation with and without intracellular nanoparticles. The findings are used to devise a strategy to quantify targeted thermal dose in a primary human liver cancer cell line. Methods Water-bath or non-invasive external RF generator (600W, 13.56 MHz) was used for hyperthermia exposures. Luciferase activity was measured using a bioluminescence assay and viability was assessed using Annexin V-FITC and Propidium iodide staining. Heat shock proteins were analyzed using western-blot analysis Results Duration-dependent luciferase denaturation was observed in SNU449 cells exposed to RF field that preceded measurable loss in viability. Loss of luciferase activity was higher in cetuximab-conjugated gold nanoparticle (C225-AuNP) treated cells. Using a standard curve from water-bath experiments, the intracellular thermal dose was calculated. Cells treated with C225-AuNP accumulated 6.07 times higher intracellular thermal dose than the untreated controls over initial 4 minutes of RF exposure. Conclusions Cancer cells when exposed to an external RF field exhibit dose-dependent protein denaturation. Luciferase denaturation assay can be used to quantify thermal dose delivered after RF exposures to cancer cells with and without nanoparticles. PMID:22515341

  14. An ancestral luciferase in the Malpighi tubules of a non-bioluminescent beetle!

    PubMed

    Viviani, V R; Prado, R A; Arnoldi, F C G; Abdalla, F C

    2009-01-01

    The evolutionary origin of beetle bioluminescence is enigmatic. Previously, weak luciferase activity was found in the non-bioluminescent larvae of Tenebrio molitor (Coleoptera: Tenebrionidae), but the detailed tissular origin and identity of the luciferase-like enzyme remained unknown. Using a closely related giant mealworm, Zophobas morio, here we show that the luciferase-like enzyme is located in the Malpighi tubules. cDNA cloning of this luciferase like enzyme, showed that it is a short AMP-ligase with weak luciferase activity which diverged long ago from beetle luciferases. The results indicate that the potential for bioluminescence in AMP-ligases is very ancient and provide a first reasonable protoluciferase model to investigate the origin and evolution of beetle luciferases. PMID:19247530

  15. Cellular Immune Response Against Firefly Luciferase After Sleeping Beauty–Mediated Gene Transfer In Vivo

    PubMed Central

    Podetz-Pedersen, Kelly M.; Vezys, Vaiva; Somia, Nikunj V.; Russell, Stephen J.

    2014-01-01

    Abstract The Sleeping Beauty (SB) transposon system has been shown to mediate new gene sequence integration resulting in long-term expression. Here the effectiveness of hyperactive SB100X transposase was tested, and we found that hydrodynamic co-delivery of a firefly luciferase transposon (pT2/CaL) along with SB100X transposase (pCMV-SB100X) resulted in remarkably sustained, high levels of luciferase expression. However, after 4 weeks there was a rapid, animal-by-animal loss of luciferase expression that was not observed in immunodeficient mice. We hypothesized that this sustained, high-level luciferase expression achieved using the SB100X transposase elicits an immune response in pT2/CaL co-administered mice, which was supported by the rapid loss of luciferase expression upon challenge of previously treated animals and in naive animals adoptively transferred with splenocytes from previously treated animals. Specificity of the immune response to luciferase was demonstrated by increased cytokine expression in splenocytes after exposure to luciferase peptide in parallel with MHC I–luciferase peptide tetramer binding. This anti-luciferase immune response observed following continuous, high-level luciferase expression in vivo clearly impacts its use as an in vivo reporter. As both an immunogen and an extremely sensitive reporter, luciferase is also a useful model system for the study of immune responses following in vivo gene transfer and expression. PMID:25093708

  16. Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution.

    PubMed

    Yasunaga, Mayu; Nakajima, Yoshihiro; Ohmiya, Yoshihiro

    2014-09-01

    Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution. PMID:25015042

  17. Structure, Mechanism, and Mutation of Bacterial Luciferase.

    PubMed

    Tinikul, Ruchanok; Chaiyen, Pimchai

    2016-01-01

    : Bacterial luciferase is a flavin-dependent monooxygenase found in bioluminescent bacteria. The enzyme catalyzes a light-emitting reaction by using reduced flavin, long chain aldehyde, and oxygen as substrates and yields oxidized flavin, carboxylic acid, and water as products with concomitant emission of blue-green light around 485-490 nm. The enzyme is a heterodimer consisting of two homologous subunits, designated as the α- and β-subunits. The reactive reaction center is located in the α-subunit, whereas the β-subunit is required for maintaining the active conformation of the α-subunit. The enzyme reaction occurs through the generation of a reactive C4a-oxygenflavin adduct, presumably C4a-peroxyflavin, before the light-emitting species is generated from the decomposition of an adduct between the C4a-peroxyflavin and the aldehyde. Because the luciferase reaction generates light, the enzyme has the potential to be used as a bioreporter for a wide variety of applications. With the recent invention of the fusion enzyme that can be expressed in mammalian cells, future possibilities for the development of additional bioreporter applications are promising. PMID:25487767

  18. Luciferase fragment complementation imaging in preclinical cancer studies

    PubMed Central

    Lake, Madryn C.; Aboagye, Eric O.

    2014-01-01

    The luciferase fragment complementation assay (LFCA) enables molecular events to be non-invasively imaged in live cells in vitro and in vivo in a comparatively cheap and safe manner. It is a development of previous enzyme complementation assays in which reporter genes are split into two, individually enzymatically inactive, fragments that are able to complement one another upon interaction. This complementation can be used to externally visualize cellular activities. In recent years, the number of studies which have used LFCAs to probe questions relevant to cancer have increased, and this review summarizes the most significant and interesting of these. In particular, it focuses on work conducted on the epidermal growth factor, nuclear and chemokine receptor families, and intracellular signaling pathways, including IP3, cAMP, Akt, cMyc, NRF2 and Rho GTPases. LFCAs which have been developed to image DNA methylation and detect RNA transcripts are also discussed. PMID:25594026

  19. Labor-effective manipulation of marine and beetle luciferases for bioassays.

    PubMed

    Kim, Sung Bae

    2012-06-01

    Engineering of luciferases with designed properties and functionalities collects great interest in bioassays. However, such an engineering including mutagenesis accompanies great consumption of time-and-labor. Here, I review an empirical approach to efficiently manipulate marine and beetle luciferases for bioassays, where a putative active site of luciferases is initially assigned with an in silico analysis, prior to the practical engineering, e.g. a hydrophilicity search reveals a characteristic hydrophilic region of luciferases as an engineering target. Amino acids in the hydrophilic region are recommended for a mutagenesis target to generate superluminescent variants of marine luciferases with prolonged bioluminescence. Empirical data suggest that a consecutive fragmentation to the assigned hydrophilic site greatly reduces time-and-labors on construction of single-chain probes. This review summarizes how to relieve the efforts for fabricating single-chain probes and potent variants of luciferases with excellent optical properties. PMID:22514115

  20. A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

    PubMed

    Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

    2015-10-01

    During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. PMID:25926684

  1. Comparative spectrochronography of different types of luciferases

    NASA Astrophysics Data System (ADS)

    Cherednikova, E. Y.; Chikishev, Andrey Y.; Dement'eva, E. I.; Kosobokova, O. V.

    1999-02-01

    We investigated the dynamic properties of two firefly luciferases: Luciola Mingrelica, that contains the only tryptophan residue and Photinus Pyralis, that contains two tryptophan residues by means of fluorescence spectrochronography method. Relaxation time of protein matrix for Luciola mingrelica is estimated to be 2 ns. The dynamic properties of luciferases differ in spite of similar composition. We investigated also the influence of microenvironment on spectral and kinetic properties of luciferin. Fluorescence decay curves and stationary spectra were measured in 7 different solvents and in complex with luciferase. The closest coincidence of decay curves in the solvents with the decay curve in the complex with luciferase was obtained in water. It means that microenvironment of luciferase is not hydrophobic, as it had been determined earlier.

  2. Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

    PubMed

    Ho, Pei-i; Yue, Kimberley; Pandey, Pramod; Breault, Lyne; Harbinski, Fred; McBride, Aaron J; Webb, Brian; Narahari, Janaki; Karassina, Natasha; Wood, Keith V; Hill, Adam; Auld, Douglas S

    2013-05-17

    Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and β-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40-70%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes. PMID:23485150

  3. Construction and characterization of a red-emitting luciferase

    NASA Astrophysics Data System (ADS)

    Eames, Brian F.; Benaron, David A.; Stevenson, David K.; Contag, Christopher H.

    1999-07-01

    Red light is transmitted through live tissue more efficiently than other wavelengths of visible light, thus by red-shifting the emission of bioluminescent reporters, we may enhance their utility for in vivo monitoring of biological processes. Codon changes at positions that may shift the yellow-green emission to red, based on studies of a related luciferase, were introduced into a variant of the North American firefly luciferase. Clones containing the desired mutation were selected based on the introduction of unique restriction enzyme sites and transfected into NIH 3T3 cells. Expression levels were evaluated using an intensified charge coupled device camera. Upon spectral analysis, all mutant luciferases demonstrated red-orange emission. Two emission peaks were detected in each spectrum, each clone with different peak heights at 560 nm and 610 nm. Sequence analyses of the compete coding region of several clones confirmed the presence of the target mutations, although sequence variation was observed at several secondary sites, likely resulting from the infidelity of Taq polymerase used in the mutagenesis protocol. A clone that demonstrated a strong 610 nm peak with a minimum shoulder at 560 nm was selected for use in animals. In summary, a red-shifted mutant of a well-characterized luciferase reporter gene was generated. Red light from this enzyme may both penetrate mammalian tissues to a greater extent and provide a tool for multicolor biological assays.

  4. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.

    PubMed

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  5. Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

    PubMed Central

    Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

    2016-01-01

    Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

  6. Mass culture of photobacteria to obtain luciferase

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Rich, E., Jr.

    1969-01-01

    Inoculating preheated trays containing nutrient agar with photobacteria provides a means for mass culture of aerobic microorganisms in order to obtain large quantities of luciferase. To determine optimum harvest time, growth can be monitored by automated light-detection instrumentation.

  7. Creation of High Efficient Firefly Luciferase

    NASA Astrophysics Data System (ADS)

    Nakatsu, Toru

    Firefly emits visible yellow-green light. The bioluminescence reaction is carried out by the enzyme luciferase. The bioluminescence of luciferase is widely used as an excellent tool for monitoring gene expression, the measurement of the amount of ATP and in vivo imaging. Recently a study of the cancer metastasis is carried out by in vivo luminescence imaging system, because luminescence imaging is less toxic and more useful for long-term assay than fluorescence imaging by GFP. However the luminescence is much dimmer than fluorescence. Then bioluminescence imaging in living organisms demands the high efficient luciferase which emits near infrared lights or enhances the emission intensity. Here I introduce an idea for creating the high efficient luciferase based on the crystal structure.

  8. Tracking angiogenesis induced by skin wounding and contact hypersensitivity using a Vegfr2-luciferase transgenic mouse.

    PubMed

    Zhang, Ning; Fang, Zuxu; Contag, Pamela R; Purchio, Anthony F; West, David B

    2004-01-15

    The vascular endothelial growth factor-2 (VEGFR2) gene is transcriptionally regulated during angiogenesis. The ability to monitor and quantify VEGFR2 expression in vivo may facilitate a better understanding of the role of VEGFR2 in different states. Here we describe a transgenic mouse, Vegfr2-luc, in which a luciferase reporter is under control of the murine VEGFR2 promoter. In adult mice, luciferase activity was highest in lung and uterus, intermediate in heart, skin, and kidney, and lower in other tissues. Luciferase expression in these tissues correlated with endogenous VEGFR2 mRNA expression. In a cutaneous wound-healing model, Vegfr2-luc expression was induced in the wound tissue. Histologic and immunohistochemical studies showed significant macrophage infiltration into the wound and induction of Vegfr2-luc expression in endothelial and stromal cells. Dexamethasone significantly suppressed Vegfr2-luc expression and macrophage infiltration into the wound, resulting in delayed healing and impaired angiogenesis. In a skin hypersensitivity reaction produced by treatment with oxazolone, Vegfr2-luc expression was induced in the ear. Treatment by dexamethasone markedly suppressed Vegfr2-luc expression and leukocyte infiltration in the ear and was correlated with reduced dermal edema and epidermal hyperplasia. The Vegfr2-luc model will be valuable in monitoring the ability of drugs to affect angiogenesis in vivo. PMID:14512298

  9. Whole-Body In Vivo Monitoring of Inflammatory Diseases Exploiting Human Interleukin 6-Luciferase Transgenic Mice.

    PubMed

    Hayashi, Makiko; Takai, Jun; Yu, Lei; Motohashi, Hozumi; Moriguchi, Takashi; Yamamoto, Masayuki

    2015-10-01

    Chronic inflammation underlies the pathological progression of various diseases, and thus many efforts have been made to quantitatively evaluate the inflammatory status of the diseases. In this study, we generated a highly sensitive inflammation-monitoring mouse system using a bacterial artificial chromosome (BAC) clone containing extended flanking sequences of the human interleukin 6 gene (hIL6) locus, in which the luciferase (Luc) reporter gene is integrated (hIL6-BAC-Luc). We successfully monitored lipopolysaccharide-induced systemic inflammation in various tissues of the hIL6-BAC-Luc mice using an in vivo bioluminescence imaging system. When two chronic inflammatory disease models, i.e., a genetic model of atopic dermatitis and a model of experimental autoimmune encephalomyelitis (EAE), were applied to the hIL6-BAC-Luc mice, luciferase bioluminescence was specifically detected in the atopic skin lesion and central nervous system, respectively. Moreover, the Luc activities correlated well with the disease severity. Nrf2 is a master transcription factor that regulates antioxidative and detoxification enzyme genes. Upon EAE induction, the Nrf2-deficient mice crossed with the hIL6-BAC-Luc mice exhibited enhanced neurological symptoms concomitantly with robust luciferase luminescence in the neuronal tissue. Thus, whole-body in vivo monitoring using the hIL6-BAC-Luc transgenic system (WIM-6 system) provides a new and powerful diagnostic tool for real-time in vivo monitoring of inflammatory status in multiple different disease models. PMID:26283726

  10. Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis

    PubMed Central

    Loening, Andreas Markus; Fenn, Timothy David; Gambhir, Sanjiv Sam

    2009-01-01

    Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in both cell culture experiments and small animal imaging. To accomplish this bioluminesce, the 37 KDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8), and present the first structures for any coelenterazine-using luciferase. These structures are based on high resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coe-lenteramide bound-luciferase, and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase’s accessory green fluorescent protein (RrGFP) was determined as well and shown to be highly similar to that of Aequorea GFP. PMID:17980388

  11. Formula optimization of the Jiashitang scar removal ointment and antiinflammatory compounds screening by NF-κB bioactivity-guided dual-luciferase reporter assay system.

    PubMed

    Gao, Jie; Tao, Jin; Zhang, Nannan; Liu, Yanjie; Jiang, Min; Hou, Yuanyuan; Wang, Qian; Bai, Gang

    2015-02-01

    Inflammation plays a role in scar formation; therefore, decreasing inflammation benefits scar removal. Jiashitang scar removal ointment (JST) is a commercially available traditional Chinese medicinal formulation. It is composed of extracts from Carthamus tinctorius L. (Car), Rheum officinale Baill. (Rhe), Salvia miltiorrhiza Beg. (Sal), and Panax notoginseng (Burk.) F. H. Chen (Pan), which are all herbs with potent antiinflammatory activities. Our aims are to optimize the formula of JST and to elucidate its antiinflammatory active components. Response surface methodology was applied to optimize proportions of the four herb extracts. The antiinflammatory effects were evaluated using in vitro and in vivo models. To screen for active components in this formula, a bioactivity-based ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis was performed. After optimization, the antiinflammatory effects of the new formula were significantly superior to the original one. Screening identified 13 active ingredients: a series of saffiomin, emodin, salvianolic acid, tanshinone, and triterpenoid saponin derivatives. These active ingredients were predicted to exert nuclear factor-κB inhibiting effects through MAPK, PI3K/AKT, and NIK-IKK pathways. In conclusion, the original formula was successfully optimized with more potent antiinflammatory activity. These methods can be applied to researches of other formulas. PMID:25363818

  12. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS

    EPA Science Inventory

    The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

  13. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS

    EPA Science Inventory

    The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

  14. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

    PubMed

    Omokoko, Tana A; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  15. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    PubMed Central

    Omokoko, Tana A.; Luxemburger, Uli; Bardissi, Shaheer; Simon, Petra; Utsch, Magdalena; Breitkreuz, Andrea; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies. PMID:27057556

  16. Bioanalytical systems based on bioluminescence resonance energy transfer using firefly luciferase.

    PubMed

    Smirnova, Darya V; Ugarova, Natalia N

    2015-01-01

    Bioanalytical systems based on the Bioluminescence Resonance Energy Transfer (BRET) are widely used in fundamental biochemical studies, as well as for screening and analysis of biologically active compounds. The Renilla luciferase is the most often used energy donor in this system despite the fact that it has low bioluminescence quantum yield and demonstrates not so stable luminescence in time as the firefly luciferase. Moreover, the bioluminescence λmax is observed in the green region of the spectrum, which complicates signal recording in tissues during in vivo experiments. The firefly luciferases do not have such drawbacks and show great promise for applications in BRET systems. Different versions of BRET systems based on firefly luciferases and the methods for increasing their efficiency are considered in this review; examples of the use of BRET systems based on the firefly luciferases for highly sensitive determination of proteases and for homogeneous immunoassays are presented. PMID:26377546

  17. Folding of firefly luciferase during translation in a cell-free system.

    PubMed Central

    Kolb, V A; Makeyev, E V; Spirin, A S

    1994-01-01

    In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell-free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell-free system while this protein is being translated from its mRNA. It is shown that ribosome-bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half-time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome. Images PMID:8062837

  18. Disturbance of firefly luciferase-based bioassays by different aluminum species.

    PubMed

    Lehmann, Caroline; Sieg, Holger; Lampen, Alfonso; Braeuning, Albert

    2016-07-01

    Luciferase-dependent assays, important for biochemical analyses of cytotoxicity and reporter genes, may be perturbed by compounds interfering with the luciferase reaction. We analyzed the impact of different aluminum (Al) species on a luciferase-based assay for determination of cellular adenosine triphosphate. Al(0) nanoparticles (Al(0)-NPs) but not Al2O3-NPs decreased luminescence, correlated to high absorbance of Al(0)-NPs. By contrast, Al ions increased the luminescent signal. Data demonstrate that luciferase-dependent assays can be reciprocally disturbed by Al-NPs or Al ions in a specific manner, depending on the particular Al species. Careful interpretation of data from such experiments is essential in order to obtain conclusive results. PMID:27059752

  19. Development and Characterization of a Human Reporter Cell Line for the Assessment of Thyroid Receptor Transcriptional Activity: A Case of Organotin Endocrine Disruptors.

    PubMed

    Illés, Peter; Brtko, Július; Dvořák, Zdeněk

    2015-08-12

    We developed and characterized the human luciferase reporter cell line PZ-TR for the assessment of thyroid receptor (TR) transcriptional activity. Triiodothyronine (T3) induced luciferase activity in a dose-dependent manner, and the sensitivity of assay allowed for the detection of nanomolar T3 concentrations. The luciferase activity was induced by a maximum of (2.42 ± 0.14)-(2.73 ± 0.23)-fold after 24 h of exposure to 10 nM T3. We did not observe a nonspecific induction of luciferase activity by other steroid hormones and VDR ligands, with the exception of partial activation by retinoic acids. Cryopreservation of PZ-TR cells did not influence their functionality, responsivity to T3, or cell morphology, even after long-term cultivation. PZ-TR cells were used to evaluate the effects of organic tin compounds on TR. We found that the tributyltin and triphenyltin derivatives induced luciferase activity and that application of organotins in combination with T3 enhanced the effect of T3. These findings indicate that organic tin compounds have potential to interfere with TR-mediated regulation of gene expression and influence the physiological activity of thyroid hormones. PMID:26208032

  20. LANSCE Activity Report

    SciTech Connect

    Amy Robinson; Audrey Archuleta; Barbara Maes; Dan Strottman; Earl Hoffman; Garth Tietjen; Gene Farnum; Geoff Greene; Joyce Roberts; Ken Johnson; Paul Lewis; Roger Pynn; Stan Schriber; Steve Sterbenz; Steve Wender; Sue Harper

    1999-02-01

    The Los Alamos Neutron Science Center Activity Report describes scientific and technological progress and achievements in LANSCE Division during the period of 1995 to 1998. This report includes a message from the Division Director, an overview of LANSCE, sponsor overviews, research highlights, advanced projects and facility upgrades achievements, experimental and user program accomplishments, news and events, and a list of publications. The research highlights cover the areas of condensed-matter science and engineering, accelerator science, nuclear science, and radiography. This report also contains a compact disk that includes an overview, the Activity Report itself, LANSCE operations progress reports for 1996 and 1997, experiment reports from LANSCE users, as well as a search capability.

  1. A Luciferase-Based Quick Potency Assay to Predict Chondrogenic Differentiation.

    PubMed

    Oberbauer, Eleni; Steffenhagen, Carolin; Feichtinger, Georg; Hildner, Florian; Hacobian, Ara; Danzer, Martin; Gabriel, Christian; Redl, Heinz; Wolbank, Susanne

    2016-05-01

    Chondrogenic differentiation of adipose-derived stem cells (ASC) is challenging but highly promising for cartilage repair. Large donor variability of chondrogenic differentiation potential raises the risk for transplantation of cells with reduced efficacy and a low chondrogenic potential. Therefore, quick potency assays are required to control the potency of the isolated cells before cell transplantation. Current in vitro methods to analyze the differentiation capacity are time-consuming, and thus, a novel enhancer and tissue-specific promoter combination was used for the detection of chondrogenic differentiation of ASC in a novel quick potency bioassay. Human primary ASC were cotransfected with the Metridia luciferase-based collagen type II reporter gene pCMVE_ACDCII-MetLuc together with a Renilla control plasmid and analyzed for their chondrogenic potential. On day 3 after chondrogenic induction, the luciferase activity was induced in all tested donors under three-dimensional culture conditions and, in a second approach, also under two-dimensional (2D) culture conditions. With our newly developed quick potency bioassay, we can determine chondrogenic potential already after 3 days of chondrogenic induction and under 2D culture conditions. This will enhance the efficiency of testing cell functionality, which should allow in the future to predict the suitability of cells derived from individual patients for cell therapies in a very short time and at low costs. PMID:27019357

  2. Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications

    PubMed Central

    Hunt, Eric A.; Moutsiopoulou, Angeliki; Ioannou, Stephanie; Ahern, Katelyn; Woodward, Kristen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K.

    2016-01-01

    Gaussia luciferase (Gluc)—with its many favorable traits such as small size, bright emission, and exceptional stability—has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed. PMID:27271118

  3. Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications.

    PubMed

    Hunt, Eric A; Moutsiopoulou, Angeliki; Ioannou, Stephanie; Ahern, Katelyn; Woodward, Kristen; Dikici, Emre; Daunert, Sylvia; Deo, Sapna K

    2016-01-01

    Gaussia luciferase (Gluc)-with its many favorable traits such as small size, bright emission, and exceptional stability-has become a prominent reporter protein for a wide range of bioluminescence-based detection applications. The ten internal cysteine residues crucial to functional structure formation, however, make expression of high quantities of soluble protein in bacterial systems difficult. In addition to this challenge, the current lack of structural data further complicates the use of Gluc for in vitro applications, such as biosensors, or cellular delivery, both of which rely heavily on robust and reproducible bioconjugation techniques. While Gluc is already appreciably small for a luciferase, a reduction in size that still retains significant bioluminescent activity, in conjunction with a more reproducible bioorthogonal method of chemical modification and facile expression in bacteria, would be very beneficial in biosensor design and cellular transport studies. We have developed truncated variants of Gluc, which maintain attractive bioluminescent features, and have characterized their spectral and kinetic properties. These variants were purified in high quantities from a bacterial system. Additionally, a C-terminal linker has been incorporated into these variants that can be used for reliable, specific modification through tyrosine-based bioconjugation techniques, which leave the sensitive network of cysteine residues undisturbed. PMID:27271118

  4. Genetic modification in organ transplantation and in vivo luciferase imaging

    NASA Astrophysics Data System (ADS)

    Murakami, Takashi; Inoue, Sei-ichiro; Sato, Yuki; Ajiki, Takashi; Ohsawa, Ichiro; Kobayashi, Eiji

    2005-04-01

    The genetic modification for organ transplantation is one of the most promising strategies to regulate allogeneic immune response. Organ-selective gene transfer has especially benefit to control local immune responses. Based on the catheter technique, we tested to deliver naked plasmid DNA to target graft organs of rats (liver and limbs) by a rapid injection (hydrodynamics-based transfection). Recent advances in transplantation have been achieved by visualization of cellular process and delivered gene expression during the inflammatory process by using non-invasive in vivo imaging. Herein, we examined the fate of genetically modified grafts using a firefly luciferase expression plasmid. For liver modification before transplantation, 6.25% of body weight PBS containing plasmid DNA was injected into the liver through the inferior vena cava using a catheter, and the liver was subsequently transplanted to the recipient rat. For limb modification, the femoral caudal epigastric vein was used. In the rat liver transplantation model, substantial luciferase expression was visualized and sustained for only a few days in the grafted liver. We also addressed stress responses by this hydrodynamics procedure using reporter plasmids containing cis-acting enhancer binding site such as NF-kappa B, cAMP, or heat shock response element. In contrast to hepatic transduction, this genetic limb targeting achieved long lasting luciferase expression in the muscle for 2 months or more. Thus, our results suggest that this catheter-based in vivo transfection technique provides an effective strategy for organ-selective gene modification in transplantation, and the bioluminescent imaging is broadening its potential for evaluation to various preclinical studies.

  5. Using luciferase to image bacterial infections in mice.

    PubMed

    Chang, Mi Hee; Cirillo, Suat L G; Cirillo, Jeffrey D

    2011-01-01

    Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load. This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of

  6. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    PubMed Central

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-01-01

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 Å cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the “off-target” effect of a small molecule is mediated by an MAI mechanism. PMID:20194791

  7. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  8. Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coli*

    PubMed Central

    Campbell, Zachary T.; Baldwin, Thomas O.

    2009-01-01

    Unlike the vast majority of flavoenzymes, bacterial luciferase requires an exogenous source of reduced flavin mononucleotide for bioluminescence activity. Within bioluminescent bacterial cells, species-specific oxidoreductases are believed to provide reduced flavin for luciferase activity. The source of reduced flavin in Escherichia coli-expressing bioluminescence is not known. There are two candidate proteins potentially involved in this process in E. coli, a homolog of the Vibrio harveyi Frp oxidoreductase, NfsA, and a luxG type oxidoreductase, Fre. Using single gene knock-out strains, we show that deletion of fre decreased light output by greater than two orders of magnitude, yet had no effect on luciferase expression in E. coli. Purified Fre is capable of supporting bioluminescence in vitro with activity comparable to that with the endogenous V. harveyi reductase (Frp), using either FMN or riboflavin as substrate. In a pull-down experiment, we found that neither Fre nor Frp co-purify with luciferase. In contrast to prior work, we find no evidence for stable complex formation between luciferase and oxidoreductase. We conclude that in E. coli, an enzyme primarily responsible for riboflavin reduction (Fre) can also be utilized to support high levels of bioluminescence. PMID:19139094

  9. Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit.

    PubMed

    Xin, X; Xi, L; Tu, S C

    1991-11-26

    The available sequences for the different bacterial luciferases reveal five conserved histidyl residues at positions 44, 45, 82, 224, and 285 of the alpha subunit. Ten variants of Vibrio harveyi luciferase were obtained by selective site-directed mutations of these five histidines. The essentiality of alpha His44 and alpha His45 was indicated by 4-7 orders of magnitude of bioluminescence activity reductions resulting from the substitution of either histidine by alanine (alpha H44A or alpha H45A), aspartate (alpha H44D or alpha H45D), or lysine (alpha H45K). Moreover, alpha H44A and alpha H45A were distinct from the native luciferase in thermal stabilities. Mutations at the other three positions also resulted in activity reductions ranging from a fewfold to 3 orders of magnitude. Despite these widely different bioluminescence light outputs, mutated luciferases exhibited, in nonturnover in vitro assays, light emission decay rates mostly similar to that of the native luciferase using octanal, decanal, or dodecanal as a substrate. This is attributed to a similarity in the catalytic rate constants of the light-emitting pathway for the native and mutated luciferases, but various mutated luciferases suffer in different degrees from competing dark reaction(s). In accord with this interpretation, the bioluminescence activities of mutated luciferases showed a general parallel with the relative stabilities of their 4a-hydroperoxyflavin intermediate species. Furthermore, the drastically reduced bioluminescence activities for luciferases with the alpha His44 or alpha His45 substituted by aspartate, alanine, or lysine were accompanied by little or no activities for consuming the aldehyde substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1958663

  10. Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

    PubMed

    Adams, Spencer T; Mofford, David M; Reddy, G S Kiran Kumar; Miller, Stephen C

    2016-04-11

    Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno-associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal. PMID:26991209

  11. How to Fabricate Functional Artificial Luciferases for Bioassays.

    PubMed

    Kim, Sung-Bae; Fujii, Rika

    2016-01-01

    The present protocol introduces fabrication of artificial luciferases (ALuc(®)) by extracting the consensus amino acids from the alignment of copepod luciferase sequences. The made ALucs have unique sequential identities that are phylogenetically distinctive from those of any existing copepod luciferase. Some ALucs exhibited heat stability, and strong and greatly prolonged optical intensities. The made ALucs are applicable to various bioassays as an optical readout, including live cell imaging, single-chain probes, and bioluminescent tags of antibodies. The present protocol guides on how to fabricate a unique artificial luciferase with designed optical properties and functionalities. PMID:27424894

  12. Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.

    PubMed

    Tang, Yan-Dong; Liu, Ji-Ting; Fang, Qiong-Qiong; Wang, Tong-Yun; Sun, Ming-Xia; An, Tong-Qing; Tian, Zhi-Jun; Cai, Xue-Hui

    2016-04-01

    A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures. PMID:27043610

  13. Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds

    PubMed Central

    Tang, Yan-Dong; Liu, Ji-Ting; Fang, Qiong-Qiong; Wang, Tong-Yun; Sun, Ming-Xia; An, Tong-Qing; Tian, Zhi-Jun; Cai, Xue-Hui

    2016-01-01

    A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures. PMID:27043610

  14. Low-cost system for real-time monitoring of luciferase gene expression.

    PubMed

    Gailey, P C; Miller, E J; Griffin, G D

    1997-03-01

    In some mammalian cells transfected with luciferase reporter genes, the luciferase/luciferin reaction in a cell monolayer produces a very small light flux. While the low light levels are often measurable with single-photon counting cameras, these devices are expensive and may require long averaging times to acquire an image. We describe an approach for real-time monitoring of light produced by luciferase gene expression in intact, cultured cells using readily available and relatively inexpensive components. The system uses a single-photon counting photomultiplier tube with built-in high voltage supply and photon counting circuitry to rapidly measure average light output from growing cells in a 35 mm culture dish. The fast, accurate and highly sensitive response of the system makes it useful for studying the dynamics of gene expression over time periods ranging from minutes to days. PMID:9067033

  15. Quantum/molecular mechanics study of firefly bioluminescence on luciferase oxidative conformation

    NASA Astrophysics Data System (ADS)

    Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

    2014-07-01

    This is the first report of a computational study of the color tuning mechanism of firefly bioluminescence, using the oxidative conformation of luciferase. The results of these calculations demonstrated that the electrostatic field generated by luciferase is fundamental both for the emission shift and efficiency. Further calculations indicated that a shift in emission is achieved by modulating the energy, at different degrees, of the emissive and ground states. These differences in energy modulation will then lead to changes in the energy gap between the states.

  16. Use of Luciferase Chimaera to Monitor PLCζ Expression in Mouse Eggs

    NASA Astrophysics Data System (ADS)

    Swann, Karl; Campbell, Karen; Yu, Yuansong; Saunders, Christopher; Lai, F. Anthony

    The microinjection of cRNA encoding phospholipase Cζ (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLCζ protein that is expressed in eggs after injection of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged PLCζ with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The use of luciferase to monitor protein expression after injection of cRNA is a sensitive and effective method that efficiently allows for sets of eggs to be used for PLCζ quantitation, Ca2+ imaging, and studies of embryo development.

  17. Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

    NASA Astrophysics Data System (ADS)

    Close, Dan M.; Hahn, Ruth E.; Patterson, Stacey S.; Baek, Seung J.; Ripp, Steven A.; Sayler, Gary S.

    2011-04-01

    Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 104 cells but at the cost of increasing overall image integration time.

  18. Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

    PubMed Central

    McCabe, Mark J.; Hu, Youli; Gregory, Louise C.; Gaston-Massuet, Carles; Alatzoglou, Kyriaki S.; Saldanha, José W.; Gualtieri, Angelica; Thankamony, Ajay; Hughes, Ieuan; Townshend, Sharron; Martinez-Barbera, Juan-Pedro; Bouloux, Pierre-Marc; Dattani, Mehul T.

    2015-01-01

    KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD. Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter. The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)]. Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene. PMID:26375424

  19. STUDIES ON BIOLUMINESCENCE : IX. CHEMICAL NATURE OF CYPRIDINA LUCIFERIN AND CYPRIDINA LUCIFERASE.

    PubMed

    Harvey, E N

    1919-01-20

    There seems to be very little doubt but that luciferase is a protein or so closely associated with proteins that their removal destroys its characteristic properties. The particular group of proteins to which it belongs may be arrived at by a process of exclusion, and only the group of albumins has properties which agree completely with those of luciferase. Dubois believes Pholas luciferase to be an oxidizing enzyme similar to the oxydones of Battelli and Stern because it is readily destroyed by fat solvents such as chloroform, strong alcohol, etc. He has detected iron in a luciferase solution which has dialyzed against running water for a long time, and believes it to be made up of protein in combination with iron and to act as an "oxyzymase ferrique." Cypridina luciferase, on the other hand, is not readily destroyed by fat solvents. Toluene and chloroform are good preservatives, and I often make use of them for this purpose, keeping the luciferase solutions for many months. Professor A. H. Phillips of Princeton University has very kindly analyzed some whole dried Cypridinoe for me and finds iron, copper, and manganese but no zinc or vanadium to be present. Whether these metals are connected with the action of Cypridina luciferase is uncertain, but it is significant that all three of the metals thought to be concerned in organic oxidations are present. Although a large amount of luciferin mixed with a small amount of luciferase will use up all the latter, I agree with Dubois that luciferase has sufficient properties in common with the enzymes as a class to be considered an enzyme. The peroxidases are well known to be used up in the reactions they accelerate. All workers on enzymes agree that the more enzymes are purified the less active they become. The chemical procedures necessary to remove foreign material bring about irreversible changes in the enzyme itself, a characteristic also of many protein groups and of the colloidal state in general. This is true in

  20. Cell-free production of Gaussia princeps luciferase – antibody fragment bioconjugates for ex vivo detection of tumor cells

    PubMed Central

    Patel, Kedar G.; Ng, Patrick P.; Kuo, Chiung-Chi; Levy, Shoshana; Levy, Ronald; Swartz, James R.

    2016-01-01

    Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc–scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide–alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA–tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc–scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker. PMID:19852937

  1. Immobilization of Firefly Luciferase on PVA-co-PE Nanofibers Membrane as Biosensor for Bioluminescent Detection of ATP.

    PubMed

    Wang, Wenwen; Zhao, Qinghua; Luo, Mengying; Li, Mufang; Wang, Dong; Wang, Yuedan; Liu, Qiongzhen

    2015-09-16

    The bioluminescent reaction catalyzed by firefly luciferase has become widely established as an outstanding analytical system for assay of adenosine triphosphate (ATP). When in solution, the luciferase is unstable and cannot be reused. The problem can be partially solved by immobilizing the luciferase on solid substrates. The poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibers membrane has abundant active hydroxyl groups on the surface. The PVA-co-PE nanofibers membrane was first activated by cyanuric chloride with triazinyl group. Then the activated PVA-co-PE nanofibers membrane was subsequently reacted with 1,3-propanediamine and biotin. The firefly luciferase was immobilized onto the surface of 1,3-propanediamine- and biotin-functionalized membranes. The surface chemical structure and morphologies of nanofibers membranes were characterized by FTIR-ATR spectra and SEM. The hydrophilicity of membranes was tested by water contact angle measurements. The detection of fluorescence intensity displayed that the firefly-luciferase-immobilized PVA-co-PE nanofibers membranes indicated high catalytic activity and efficiency. Especially, the firefly-luciferase-immobilized nanofiber membrane which was functionalized by biotin can be a promising candidate as biosensor for bioluminescent detection of ATP because of its high detection sensitivity. PMID:26275118

  2. Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence

    NASA Astrophysics Data System (ADS)

    Wang, Yu; Akiyama, Hidefumi; Terakado, Kanako; Nakatsu, Toru

    2013-08-01

    Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.

  3. A transgenic rat with ubiquitous expression of firefly luciferase gene

    NASA Astrophysics Data System (ADS)

    Hakamata, Yoji; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    In vivo imaging strategies provide cellular and molecular events in real time that helps us to understand biological processes in living animals. The development of molecular tags such as green fluorescent proteins and luciferase from the firefly Photinus pyralis has lead to a revolution in the visualization of complex biochemical processes. We developed a novel inbred transgenic rat strain containing firefly luciferase based on the transgenic (Tg) technique in rats. This Tg rat expressed the luciferase gene ubiquitously under control of the ROSA26 promoter. Cellular immune responsiveness against the luciferase protein was evaluated using conventional skin grafting and resulted in the long-term acceptance of Tg rat skin on wild-type rats. Strikingly, organ transplant with heart and small bowel demonstrated organ viability and graft survival, suggesting that cells from luciferase-Tg are transplantable to track their fate. Taking advantage of the less immunogenic luciferase, we also tested the role of hepatocyte-infusion in a liver injury model, and bone marrow-derived cells in a skin defect model. Employed in conjunction with modern advances in optical imaging, this luciferase-Tg rat system provides an innovative animal tool and a new means of facilitating biomedical research such as in the case of regeneration medicine.

  4. Generation of a Gaussia luciferase-expressing endotheliotropic cytomegalovirus for screening approaches and mutant analyses.

    PubMed

    Falk, Jessica J; Laib Sampaio, Kerstin; Stegmann, Cora; Lieber, Diana; Kropff, Barbara; Mach, Michael; Sinzger, Christian

    2016-09-01

    For many questions in human cytomegalovirus (HCMV) research, assays are desired that allow robust and fast quantification of infection efficiencies under high-throughput conditions. The secreted Gaussia luciferase has been demonstrated as a suitable reporter in the context of a fibroblast-adapted HCMV strain, which however is greatly restricted in the number of cell types to which it can be applied. We inserted the Gaussia luciferase expression cassette into the BAC-cloned virus strain TB40-BAC4, which displays the natural broad cell tropism of HCMV and hence allows application to screening approaches in a variety of cell types including fibroblasts, epithelial, and endothelial cells. Here, we applied the reporter virus TB40-BAC4-IE-GLuc to identify mouse hybridoma clones that preferentially neutralize infection of endothelial cells. In addition, as the Gaussia luciferase is secreted into culture supernatants from infected cells it allows kinetic analyses in living cultures. This can speed up and facilitate phenotypic characterization of BAC-cloned mutants. For example, we analyzed a UL74 stop-mutant of TB40-BAC4-IE-GLuc immediately after reconstitution in transfected cultures and found the increase of luciferase delayed and reduced as compared to wild type. Phenotypic monitoring directly in transfected cultures can minimize the risk of compensating mutations that might occur with extended passaging. PMID:27326666

  5. Luciferase-dependent oxygen consumption by bioluminescent vibrios

    SciTech Connect

    Makemson, J.C.

    1986-02-01

    Oxygen uptake due to luciferase in two luminous Vibrio species was estimated in vivo by utilizing inhibitors having specificities for luciferase (decanol) and cytochromes (cyanide). Cyanide titration of respiration revealed a component of oxygen uptake less sensitive to cyanide which was completely inhibitable by low concentrations of decanol. From this it was estimated that in vivo luciferase is responsible for less than 12% (Vibrio harveyi) or 20% (Vibrio fischeri) of the total respiration. From these data in vivo bioluminescent quantum yields are estimated to be not lower than 1.7 and 2.6%, respectively.

  6. Monitoring Tumorigenesis and Senescence In Vivo with a p16INK4a-Luciferase Model

    PubMed Central

    Burd, Christin E.; Sorrentino, Jessica A.; Clark, Kelly S.; Darr, David B.; Krishnamurthy, Janakiraman; Deal, Allison M.; Bardeesy, Nabeel; Castrillon, Diego H.; Beach, David H.; Sharpless, Norman E.

    2013-01-01

    SUMMARY Monitoring cancer and aging in vivo remains experimentally challenging. Here, we describe a luciferase knockin mouse (p16LUC), which faithfully reports expression of p16INK4a, a tumor suppressor and aging biomarker. Lifelong assessment of luminescence in p16+/LUC mice revealed an exponential increase with aging, which was highly variable in a cohort of contemporaneously housed, syngeneic mice. Expression of p16INK4a with aging did not predict cancer development, suggesting that the accumulation of senescent cells is not a principal determinant of cancer-related death. In 14 of 14 tested tumor models, expression of p16LUC was focally activated by early neoplastic events, enabling visualization of tumors with sensitivity exceeding other imaging modalities. Activation of p16INK4a was noted in the emerging neoplasm and surrounding stromal cells. This work suggests that p16INK4a activation is a characteristic of all emerging cancers, making the p16LUC allele a sensitive, unbiased reporter of neoplastic transformation. PMID:23332765

  7. High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria

    PubMed Central

    2016-01-01

    In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12–13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages. PMID:27275010

  8. Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

    PubMed

    Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-01-01

    Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors. PMID:26530405

  9. ALS Activity Report 2004

    SciTech Connect

    Tamura , Lori S.

    2005-07-11

    This annual report of the Advanced Light Source details science highlights and facility developments during the year. It also offers information on events sponsored by the facility, technical specifications, and staff and publication information.

  10. Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor-reporter gene assays.

    PubMed

    Balaguer, P; Joyeux, A; Denison, M S; Vincent, R; Gillesby, B E; Zacharewski, T

    1996-02-01

    In vitro recombinant receptor-reporter gene assays have been used to assess and rank the potency of chemicals and complex mixtures suspected of possessing estrogen and (or) aryl hydrocarbon receptor (AhR) mediated activity. The environmental estrogen (E2) bioassay consists of a Gal4-human estrogen receptor chimeric construct (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc) that have been stably integrated into HeLa cells. The assay exhibits 10-fold induction in luciferase reporter gene activity following treatment with 1 nM 17 beta-estradiol and has a detection limit of approximately 5 pg of 17 beta-estradiol/mL. The AhR bioassay uses Hepa 1c1c7 wild-type cells transiently transfected with a dioxin response element regulated luciferase reporter gene. These assays were used to assess the estrogen and dioxin-like activities of naringenin, atrazine, and simazine and complex mixtures such as pulp and paper mill black liquor and urban air particulates. The activities of these chemicals and complex mixtures are confirmed using the pure antiestrogen ICI 164,384 and in in vitro gel retardation assays. Results of this study demonstrate the utility of in vitro recombinant receptor-reporter gene assays in identifying and assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures. PMID:8723035

  11. Dynamic Analysis of GH Receptor Conformational Changes by Split Luciferase Complementation

    PubMed Central

    Liu, Ying; Berry, Philip A.; Zhang, Yue; Jiang, Jing; Lobie, Peter E.; Paulmurugan, Ramasamy; Langenheim, John F.; Chen, Wen Y.; Zinn, Kurt R.

    2014-01-01

    The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharmacological manipulation, genetic alteration of JAK2 levels, and truncation of the GHR intracellular domain (ICD) tail suggested that GH acutely enhances proximity of the GHR homodimer partners independent of the presence of JAK2, phosphorylation of GHR-luciferase chimeras, or an intact ICD. However, subsequent reduction of complementation requires JAK2 kinase activity and the ICD tail. This conclusion is in contrast to existing models of the GHR activation process. PMID:25188449

  12. ESU Activity Report 2011

    ERIC Educational Resources Information Center

    Pall, Allan, Ed.; Ufert, Karina, Ed.; Slegers, Marianne, Ed.

    2011-01-01

    European Students' Union (ESU) has been representing students since 1982. In these, almost 30 years, ESU has been going from strength to strength as a European organisation. Each year has been crucial. This report brings to the fore its notable achievements and work done in 2011. The carousel of higher education reform on the national level was at…

  13. ESU Activity Report 2009

    ERIC Educational Resources Information Center

    European Students' Union (NJ1), 2009

    2009-01-01

    This report tells a short story of European Students' Union (ESU's) work in 2009. It highlights some of the key areas that ESU worked on, while giving some examples of each general area as outlined in the Plan of Work adopted by the ESU members--National Unions of Students--in November 2008. Members have linked to relevant web documents and…

  14. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

    PubMed Central

    Johnson, Barbara W.; Olson, Ken E.; Allen-Miura, Tanya; Rayms-Keller, Alfredo; Carlson, Jonathan O.; Coates, Craig J.; Jasinskiene, Nijole; James, Anthony A.; Beaty, Barry J.; Higgs, Stephen

    1999-01-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  15. Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA.

    PubMed

    Johnson, B W; Olson, K E; Allen-Miura, T; Rayms-Keller, A; Carlson, J O; Coates, C J; Jasinskiene, N; James, A A; Beaty, B J; Higgs, S

    1999-11-01

    A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3'2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5' end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo. PMID:10557332

  16. NSLS 2009 Activity Report

    SciTech Connect

    Nasta K.; Mona R.

    2009-05-01

    2009 was an incredibly exciting year for light sources at Brookhaven. The National Synchrotron Light Source (NSLS) hosted more than 2,200 visiting researchers, who, along with the about 50 members of our scientific staff, produced a total of 957 publications - about 20 percent of which appeared in premier journals. Covering topics ranging from Alzheimer's disease detection to ethanol-powered fuel cells, a sampling of these findings can be found in this Activity Report. We've also seen the resurfacing of some of our long-time users hard work. I was very proud to hear that two of the three recipients of the 2009 Nobel Prize in Chemistry have ties to the NSLS. Venki Ramakrishnan, a former employee in Brookhaven's biology department and long-time user of the NSLS, now at Cambridge University, and Thomas A. Steitz of Yale University, also a long-time NSLS user, shared the prize with Ada E. Yonath of the Weizmann Institute of Science for their work on the structure and function of the ribosome. In the late 1990s, Ramakrishnan and Steitz used protein crystallography at the NSLS to gather atomic-level images of two ribosome subunits: 30S (Ramakrishnan) and 50S (Steitz). Both laureates solved the high-resolution structures for these subunits based on this data. After struggling with a rough budget for several years, we received excellent funding, and then some, this year. In addition to NSLS operations funding, we received $3 million in funds from the American Recovery and Reinvestment Act (ARRA). We used that additional money for two exciting projects: construction of a full-field x-ray microscope and acquisition of several advanced x-ray detectors. The x-ray microscope will be able to image objects with a targeted spatial resolution of 30 nanometers. This capability will be particularly important for new initiatives in energy research and will prepare our users for the projected 1-nanometer resolution benchmark at the National Synchrotron Light Source II (NSLS-II). The

  17. Complex Regulation Pattern of IRF3 Activation Revealed by a Novel Dimerization Reporter System.

    PubMed

    Wang, Zining; Ji, Jingyun; Peng, Di; Ma, Feng; Cheng, Genhong; Qin, F Xiao-Feng

    2016-05-15

    Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in antibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in response to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type-specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-α/β receptor-mediated feed-forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation. PMID:27045107

  18. A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties.

    PubMed

    Viviani, Vadim R; Amaral, Danilo; Prado, Rogilene; Arnoldi, Frederico G C

    2011-12-01

    Firefly luciferases usually produce bioluminescence in the yellow-green region, with colors in the green and yellow-orange extremes of the spectrum being less common. Several firefly luciferases have already been cloned and sequenced, and site-directed mutagenesis studies have already identified important regions and residues for bioluminescence colors. However the structural determinants and mechanisms of bioluminescence colors turned out to be elusive, mainly when comparing luciferases with a high degree of divergence. Thus comparison of more similar luciferases producing colors in the two extremes of the spectrum could be revealing. The South-American fauna of fireflies remains largely unstudied, with some unique taxa that are not found anywhere else in the world and that produce a wide range of bioluminescence colors. Among them, fireflies of the genus Amydetes are especially interesting because its taxonomical status as an independent subfamily or as a tribe is not yet solved, and because they usually produce a continuous bright blue-shifted bioluminescence. In this work we cloned the cDNA for the luciferase of the Atlantic rain forest Amydetes fanestratus firefly, which is found near Sorocaba municipality (São Paulo, Brazil). Despite showing a higher degree of identity with the South-American Cratomorphus, the European Lampyris and the Asiatic Pyrocoelia, phylogenetical analysis of the luciferase sequence support the inclusion of Amydetes as an independent subfamily. Amydetes luciferase displays one of the most blue-shifted emission spectra (λ(max) = 538 nm) among beetle luciferases, with lower pH-sensitivity and higher affinity for ATP when compared to other luciferases, making this luciferase attractive for sensitive ATP and reporter assays. PMID:21983629

  19. Dissemination Activities Report

    ERIC Educational Resources Information Center

    Barclay, Hanna; Batatia, Hudj; Bauters, Merja; Ben Ami, Zvi; Drachman, Raul; Flouris, Giorgos; Jadin, Tanja; Jalonen, Satu; Karlgren, Klas; Karpati, Andrea; Kotzinos, Dimitris; Lakkala, Minna; Lallimo, Jiri; Moen, Anne; Nygard, Kathrine; Paavola, Sami; Padiglia, Sheila; Scapolla, Marina; Sins, Patrick; Vasileva, Tania

    2008-01-01

    In the first 24 months of the project, KP-Lab members were highly dedicated to dissemination and were engaged in various dissemination activities that contributed to the prime objective of the KP-Lab dissemination efforts which is "to make the project widely known to a variety of prospective users and, at a later stage, to promote the…

  20. Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-vargeted luciferase from neuronal cells.

    PubMed

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Genz, Lara; Püchel, Gerhard P

    2015-01-01

    Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use. PMID:26389683

  1. Measurement of fluoride-induced endoplasmic reticulum stress using Gaussia luciferase.

    PubMed

    Sharma, Ramaswamy; Tsuchiya, Masahiro; Tannous, Bakhos A; Bartlett, John D

    2011-01-01

    Endoplasmic reticulum (ER) stress and its consequent activation of the unfolded protein response (UPR) signaling pathway have been implicated in several pathophysiologic disorders as well as in drug resistance to treatment of tumors. Several techniques have been devised that qualitatively and quantitatively demonstrate the presence of ER stress and the activation of the UPR; however, most of these methods cannot be used to measure ER stress in real time. Here we describe the use of cells stably transduced with a secreted reporter, Gaussia luciferase (Gluc), to measure fluoride-induced ER stress. Factors that affect ER homeostasis, such as high-dose fluoride, will cause decreased Gluc secretion that can be measured as a decrease in Gluc activity in the culture medium supernatant. Gluc catalyzes the oxidative decarboxylation of coelenterazine (CTZ) to coeleneteramide, resulting in blue bioluminescence (λ(max) 485 nm). Therefore, Gluc activity can be easily quantified by mixing a small aliquot of the medium supernatant with CTZ and measuring the resulting bioluminescence in a luminometer. Among the various reporters used so far, Gluc is regarded as the most sensitive indicator of ER stress. A second advantage for using Gluc is its ability to function in a wide pH range. This is especially useful for studying fluoride-mediated toxicity as fluoride-induced stress is enhanced under acidic conditions. Since Gluc can be measured in a noninvasive manner, it has been used in several in vitro and in vivo applications. In this chapter, we detail our methodology for using Gluc to monitor fluoride-induced ER stress. PMID:21329797

  2. Identification of agents that promote endoplasmic reticulum stress using an assay that monitors luciferase secretion

    PubMed Central

    Doudican, Nicole A.; Wen, Shih Ya; Mazumder, Amitabha; Orlow, Seth J.

    2015-01-01

    Disruption of protein processing in the secretory pathway is a measurable hallmark of endoplasmic reticulum (ER) stress. Activation of ER stress-mediated pathways has been implicated in numerous diseases including cancer. To identify agents that induce ER stress, we established a screen for compounds that reduce secretion of the reporter protein Gaussia luciferase (GLUC). Given the clinically validated importance of targeting ER stress-mediated pathways in the treatment of multiple myeloma (MM), we used this hematological malignancy as a model for validating our screening system. From a screen of 2000 marketed drugs and natural compounds in KMS11 and ARP1 MM cells, we identified 97 agents that reduced GLUC secretion in both cell lines by at least 30%. In order to confirm inducers of ER stress, we applied a secondary screen that assessed splicing of the unfolded protein response (UPR) transcription factor XBP1. One agent, theaflavin-3,3′–digallate (TF-3), was chosen based on its history of safe human consumption and further validated through studies of ER stress-related pathways including the UPR and apoptosis. Given these promising results, this screen could be a useful tool to identify agents targeting ER stress-related mechanisms in other cellular systems wherein ER stress plays a role in disease etiology. PMID:24371212

  3. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

    PubMed

    England, Christopher G; Ehlerding, Emily B; Cai, Weibo

    2016-05-18

    The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system. PMID:27045664

  4. A novel screening system based on VanX-mediated autolysis-Application to Gaussia luciferase.

    PubMed

    Wu, Nan; Kamioka, Tetsuya; Kuroda, Yutaka

    2016-07-01

    We report a novel bacterial screening protocol based on co-expressing the target protein with VanX, an enzyme which mediates Escherichia coli's autolysis and the release of the target protein into the culture medium, thereby facilitating activity measurement and screening from crude medium. This protocol as assessed with 19 Gaussia luciferase (GLuc) expressing colonies, was able to detect bioluminescence wavelength shift as small as 1.5 nm. We demonstrate the performance and versatility of this protocol by applying it to a semi-rational search for GLuc variants with red-shifted bioluminescence. Six GLuc's sites, F113, I114, W143, L144, A149, and F151, were randomly mutated, and for each site, 50 colonies were cultivated in 3 mL samples, from which bioluminescence was measured without purification. We identified two GLuc single mutation red-shifted variants: W143V and L144A. Their red shifted bioluminescence and biophysical/biochemical properties were confirmed using HPLC purified variants. Biotechnol. Bioeng. 2016;113: 1413-1420. © 2015 Wiley Periodicals, Inc. PMID:26694096

  5. E-Division activities report

    SciTech Connect

    Barschall, H.H.

    1981-07-01

    This report describes some of the activities in E (Experimental Physics) Division during the past year. E-Division carries out research and development in areas related to the missions of the Laboratory. Many of the activities are in pure and applied atomic and nuclear physics and in material science. In addition this report describes work on accelerators, microwaves, plasma diagnostics, determination of atmospheric oxygen and of nitrogen in tissue.

  6. E-Division activities report

    SciTech Connect

    Barschall, H.H.

    1983-07-01

    This report describes some of the activities in E (Experimental Physics) Division during the past year. E-division carries out research and development in areas related to the missions of the Laboratory. Many of the activities are in pure and applied atomic and nuclear physics and in materials science. In addition, this report describes development work on accelerators and on instrumentation for plasma diagnostics, nitrogen exchange rates in tissue, and breakdown in gases by microwave pulses.

  7. In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

    PubMed Central

    Sadeghi, Somayeh; Seyed, Negar; Etemadzadeh, Mohammad-Hossein; Abediankenari, Saeid; Rafati, Sima; Taheri, Tahereh

    2015-01-01

    Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency. PMID:26323836

  8. Determination of relative assay response factors for toxic chlorinated and bromiated dioxins/furans using an enyme immunoassay (EIA) and a chemically-activated luciferase gene expression cell bioassay (CALUX)

    EPA Science Inventory

    Determination of dioxin-like activity requires knowledge of both the concentration and toxicity to evaluate the risk of adverse human health and environmental effects. The dioxin-like response of several polybrominated dibenzo-p-dioxins/furans (PBDDs/Fs) and polybrominated/chlori...

  9. Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.

    PubMed

    Oyama, Hiroyuki; Morita, Izumi; Kiguchi, Yuki; Miyake, Sayaka; Moriuchi, Ayaka; Akisada, Tatsuki; Niwa, Toshifumi; Kobayashi, Norihiro

    2015-12-15

    In this study, we show the utility of Gaussia luciferase (GLuc), which is much smaller than previously found luciferases, as the fusion partner with artificial antibody species for developing sensitive immunoassay systems. As an example, we constructed a bioluminescent enzyme-linked immunosorbent assay (BL-ELISA) system determining the major glucocorticoid cortisol. A monoclonal antibody was newly elicited against a cortisol-albumin conjugate, and the genes encoding its variable domains (VH and VL) were cloned and combined to encode a single-chain Fv fragment (scFv). scFv was then linked to the wild-type GLuc gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several Escherichia coli strains. Notably, the wild-type GLuc fusion protein (scFv-wtGLuc) showed the most suitable luminescent properties for BL-ELISAs. In our system, scFv-wtGLuc was reacted competitively with the analyte and immobilized cortisol moieties, and the bound GLuc activity was monitored with coelenterazine as the substrate. Successful batch-type luminescence detection was achieved using a plate reader without built-in injectors. The midpoint and limit of detection in a typical dose-response curve were 4.1 and 0.26 pg/assay, respectively, thus exhibiting much more sensitivity than conventional cortisol immunoassays. Serum cortisol levels (as the sum with cortisone) for healthy subjects, determined without any pretreatment, were compatible with reported reference ranges. The scFv-wtGLuc probe was stable over a year under storage as periplasmic extracts at -30 °C or with repeated freeze-thawing. These results suggest that GLuc fusions with antibody fragments might serve as useful and highly sensitive immunoassay probes in various clinical settings. PMID:26625180

  10. Development of red-shifted mutants derived from luciferase of Brazilian click beetle Pyrearinus termitilluminans.

    PubMed

    Nishiguchi, Tomoki; Yamada, Toshimichi; Nasu, Yusuke; Ito, Mashiho; Yoshimura, Hideaki; Ozawa, Takeaki

    2015-10-01

    Luciferase, a bioluminescent protein, has been used as an analytical tool to visualize intracellular phenomena. Luciferase with red light emission is particularly useful for bioluminescence imaging because of its high transmittance in mammalian tissues. However, the luminescence intensity of existing luciferases with their emission over 600 nm is insufficient for imaging studies because of their weak intensities. We developed mutants of Emerald luciferase (Eluc) from Brazilian click beetle (Pyrearinus termitilluminans), which emits the strongest bioluminescence among beetle luciferases. We successively introduced four amino acid mutations into the luciferase based on a predicted structure of Eluc using homology modeling. Results showed that quadruple mutations R214K/H241K/S246H/H347A into the beetle luciferase emit luminescence with emission maximum at 626 nm, 88-nm red-shift from the wild-type luciferase. This mutant luciferase is anticipated for application in in vivo multicolor imaging in living samples. PMID:26313214

  11. Development of red-shifted mutants derived from luciferase of Brazilian click beetle Pyrearinus termitilluminans

    NASA Astrophysics Data System (ADS)

    Nishiguchi, Tomoki; Yamada, Toshimichi; Nasu, Yusuke; Ito, Mashiho; Yoshimura, Hideaki; Ozawa, Takeaki

    2015-10-01

    Luciferase, a bioluminescent protein, has been used as an analytical tool to visualize intracellular phenomena. Luciferase with red light emission is particularly useful for bioluminescence imaging because of its high transmittance in mammalian tissues. However, the luminescence intensity of existing luciferases with their emission over 600 nm is insufficient for imaging studies because of their weak intensities. We developed mutants of Emerald luciferase (Eluc) from Brazilian click beetle (Pyrearinus termitilluminans), which emits the strongest bioluminescence among beetle luciferases. We successively introduced four amino acid mutations into the luciferase based on a predicted structure of Eluc using homology modeling. Results showed that quadruple mutations R214K/H241K/S246H/H347A into the beetle luciferase emit luminescence with emission maximum at 626 nm, 88-nm red-shift from the wild-type luciferase. This mutant luciferase is anticipated for application in in vivo multicolor imaging in living samples.

  12. Luciferase-Based, High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes.

    PubMed

    Lucantoni, Leonardo; Fidock, David A; Avery, Vicky M

    2016-04-01

    The discovery of new antimalarial drugs able to target both the asexual and gametocyte stages ofPlasmodium falciparumis critical to the success of the malaria eradication campaign. We have developed and validated a robust, rapid, and cost-effective high-throughput reporter gene assay to identify compounds active against late-stage (stage IV and V) gametocytes. The assay, which is suitable for testing compound activity at incubation times up to 72 h, demonstrates excellent quality and reproducibility, with averageZ' values of 0.85 ± 0.01. We used the assay to screen more than 10,000 compounds from three chemically diverse libraries. The screening outcomes highlighted the opportunity to use collections of compounds with known activity against the asexual stages of the parasites as a starting point for gametocytocidal activity detection in order to maximize the chances of identifying gametocytocidal compounds. This assay extends the capabilities of our previously reported luciferase assay, which tested compounds against early-stage gametocytes, and opens possibilities to profile the activities of gametocytocidal compounds over the entire course of gametocytogenesis. PMID:26787698

  13. Endotoxin assay by bioluminescence using mutant firefly luciferase.

    PubMed

    Noda, Kenichi; Goto, Hitoshi; Murakami, Yuji; Ahmed, Abo Bakr F; Kuroda, Akio

    2010-02-15

    The Limulus reaction is an application of the defense mechanism of horseshoe crab for endotoxin detection. Endotoxin is a component of the cell wall in the outer membrane of gram-negative bacteria, and causes fever or shock when it enters the human blood stream. For endotoxin detection, gel formation or turbidity of the coagulation factor chromogen or fluorescence-modified peptide is used. However, these conventional methods have problems with regard to their measurement time or sensitivity. We recently obtained a mutant firefly luciferase that has a luminescence intensity over 10-fold higher than that of the wild type. Therefore, we developed a new endotoxin detection method that combines the Limulus reaction and bioluminescence using mutant luciferase. The new method detects 0.0005EU/ml of endotoxin within 15min. PMID:19850001

  14. Recombinant porcine reproductive and respiratory syndrome virus expressing luciferase genes provide a new indication of viral propagation in both permissive and target cells.

    PubMed

    Gao, Fei; Qu, Zehui; Li, Liwei; Yu, Lingxue; Jiang, Yifeng; Zhou, Yanjun; Yang, Shen; Zheng, Hao; Huang, Qinfeng; Tong, Wu; Tong, Guangzhi

    2016-08-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has a condensed single-stranded positive-sense RNA genome that contains several overlapping regions. The transcription regulatory sequence (TRS) is the important cis-acting element participating in PRRSV discontinuous transcription process. Based on reverse genetic system of type 2 highly pathogenic PRRSV cell-passage attenuated strain pHuN4-F112, firefly luciferase or Renilla luciferase genes were inserted between ORF1b and ORF2. An extra TRS6 was embedded behind the foreign luciferase genes. pA-Fluc and pA-Rluc were constructed and successfully rescued in MARC-145 cells. The phenotypical characteristics of the progeny virus were indistinguishable from those of vHuN4-F112 and were genetically stable for at least 25 cell passages. Mutant virus-infected cells were lysed at different time points to assess luciferase activities and measure foreign gene expression levels. The results showed identical variations in the luciferase activities of the recombinants in MARC-145 cells, indicating that they were suitable for monitoring viral propagation in PRRSV-permissive cell cultures. They were also used to infect pulmonary alveolar macrophages, which yielded similar variations in luciferase activities. Therefore, vA-Fluc and vA-Rluc present powerful new tools to monitor PRRSV propagation in both passaged and target cells. PMID:27473986

  15. Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

    1975-01-01

    A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

  16. A plant 35S CaMV promoter induces long-term expression of luciferase in Atlantic salmon

    PubMed Central

    Seternes, Tore; Tonheim, Tom C.; Myhr, Anne I.; Dalmo, Roy A.

    2016-01-01

    The long-term persistence and activity of a naked plasmid DNA (pGL3-35S) containing a luc gene (reporter gene) controlled by a plant 35S CaMV promoter was studied in Atlantic salmon (Salmo salar L.) after injection. Atlantic salmon (mean weight 70 grams) were injected intramuscularly with 100 μg of plasmid DNA. Blood, different tissues and organs were sampled at different time points up to day 535 after injection. Southern blot analysis suggested the presence of extra-chromosomally open circular, linear and supercoiled topoforms of pGL3-35S at day 150 after injection. At day 536 open circular and supercoiled topoforms were detected. Luciferase activity was detected at the injection site up to 536 days post-injection of pGL3-35S, where it peaked at day 150 and decreased to approximately 17% of its maximum activity by day 536. Our study demonstrated that a plasmid containing the 35S promoter was able to induce expression of a reporter gene/protein in fish in vivo and that the plasmid DNA persisted for a prolonged time after intramuscular injection. PMID:27114167

  17. A plant 35S CaMV promoter induces long-term expression of luciferase in Atlantic salmon.

    PubMed

    Seternes, Tore; Tonheim, Tom C; Myhr, Anne I; Dalmo, Roy A

    2016-01-01

    The long-term persistence and activity of a naked plasmid DNA (pGL3-35S) containing a luc gene (reporter gene) controlled by a plant 35S CaMV promoter was studied in Atlantic salmon (Salmo salar L.) after injection. Atlantic salmon (mean weight 70 grams) were injected intramuscularly with 100 μg of plasmid DNA. Blood, different tissues and organs were sampled at different time points up to day 535 after injection. Southern blot analysis suggested the presence of extra-chromosomally open circular, linear and supercoiled topoforms of pGL3-35S at day 150 after injection. At day 536 open circular and supercoiled topoforms were detected. Luciferase activity was detected at the injection site up to 536 days post-injection of pGL3-35S, where it peaked at day 150 and decreased to approximately 17% of its maximum activity by day 536. Our study demonstrated that a plasmid containing the 35S promoter was able to induce expression of a reporter gene/protein in fish in vivo and that the plasmid DNA persisted for a prolonged time after intramuscular injection. PMID:27114167

  18. von Hippel-Lindau β-domain-luciferase fusion protein as a bioluminescent hydroxyproline sensor for a hypoxia-inducible factor prolyl hydroxylase assay.

    PubMed

    Hong, Sungchae; Yum, Soohwan; Ha, Nam-Chul; Jung, Yunjin

    2010-12-15

    Hypoxia-inducible factor prolyl hydroxylases (HPHs) are responsible for hydroxylation of proline residues in hypoxia-inducible factor-α (HIF-α), resulting in von Hippel-Lindau (VHL)-mediated proteasome degradation of the hydroxylated proteins. Pharmacological inhibition of the enzyme leads to stabilization of HIF-α proteins and consequent activation of HIF, which provides therapeutic benefit for a variety of tissues undergoing ischemic stress. In an effort to develop a new assay for measuring HPH activity, we designed a fusion protein, VHL β-domain-luciferase. Recombinant fusion protein with a glutathione S-transferase (GST) tag was purified from Escherichia coli. GST-VHL β-domain-luciferase with C-terminal deletion (GVbL-CD) was obtained as a major product and found to have luciferase activity. In a GVbL-CD capture assay using HIF peptide-bound beads, at least a 13-fold increase in luciferase activity was elicited for HIF peptide with hydroxyproline compared with unhydroxylated HIF peptide. HPH inhibitory activities of known HPH inhibitors or HIF-1α inducers were assessed using this assay, whose results were in good agreement with those obtained from conventional methods. The competitive effect of 2-ketoglutarate on dimethyloxalylglycine-mediated HPH inhibition was assessed very well in the new assay. Taken together, the VHL β-domain protein with luciferase activity is of use for HPH activity assay. PMID:20705044

  19. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed.

    PubMed

    Dale, Renee; Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi; Kato, Naohiro

    2016-01-01

    The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively. PMID:26886551

  20. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed

    PubMed Central

    Dale, Renee; Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi; Kato, Naohiro

    2016-01-01

    The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively. PMID:26886551

  1. Generation of stable L. major(+EGFP-LUC) and simultaneous comparison between EGFP and luciferase sensitivity.

    PubMed

    Taheri, Tahereh; Saberi Nik, Hana; Seyed, Negar; Doustdari, Fatemeh; Etemadzadeh, Mohammad-Hossein; Torkashvand, Fatemeh; Rafati, Sima

    2015-03-01

    Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice. Through different specific tools, EGFP and LUC signals from the parasite were detectable and measurable within a mammalian host and promastigotes. Here, the LUC protein provided a higher level of sensitivity than did EGFP, so that infection was detectable at an earlier stage of the disease in the footpad (injection site) and lymph nodes by bioluminescence. These results depicted that: (1) both quantitative reporter genes, EGFP and LUC, could be simultaneously used to detect parasitemia in vitro and in vivo and (2) sensitivity of firefly luciferase was 10-fold higher than that of EGFP in promastigotes. PMID:25637784

  2. Elemental sulfur: toxicity in vivo and in vitro to bacterial luciferase, in vitro yeast alcohol dehydrogenase, and bovine liver catalase.

    PubMed

    Cetkauskaite, Anolda; Pessala, Piia; Södergren, Anders

    2004-08-01

    The aim of this research was to analyze the effects and the modes of action of elemental sulfur (S(0)) in bioluminescence and respiration of Vibrio fischeri cells and the enzymes crude luciferase, pure catalase, and alcohol dehydrogenase (ADH). Metallic copper removed sulfur and reduced the toxicity of acetone extracts of sediment samples analyzed in the bioluminescence test. The sulfur inhibition of cell bioluminescence was noncompetitive with decanal, the luciferase substrate; reversible, with maximum toxicity after 15 min (EC(50) = 11.8 microg/L); and almost totally recovered after 2 h. In vitro preincubation of crude luciferase extract with sulfur (0.28 ppm) weakly inhibited bioluminescence at 5 min, but at 30 min the inhibition reached 60%. Increasing the concentration of sulfur in the parts per million concentration range in vitro decreased bioluminescence, which was not constant, but depended on exposure time, and no dead-end/total inhibition was observed. The redox state of enzymes in the in vitro system significantly affected inhibition. Hydrogen peroxide restored fully and the reducing agent dithiothreitol, itself toxic, restored only partially luciferase activity in the presence of sulfur. Sulfur (5.5 ppm) slightly inhibited ADH and catalase, and dithiothreitol enhanced sulfur inhibition. High sulfur concentrations (2.2 ppm) inhibited the bioluminescence and enhanced the respiration rate of V. fischeri cells. Elemental sulfur data were interpreted to show that sulfur acted on at least a few V. fischeri cell sites: reversibly modifying luciferase at sites sensitive to/protected by oxidative and reducing agents and by affecting electron transport processes, resulting in enhanced oxygen consumption. Sulfur together with an enzyme reducing agent inhibited the oxidoreductive enzymes ADH and catalase, which have --SH groups, metal ion cofactors, or heme, respectively, in their active centers. PMID:15269910

  3. Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

    PubMed

    Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

    2015-11-15

    Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. PMID:26022509

  4. Tracking of dendritic cell migration into lymph nodes using molecular imaging with sodium iodide symporter and enhanced firefly luciferase genes

    PubMed Central

    Lee, Ho Won; Yoon, Seung Yun; Singh, Thoudam Debraj; Choi, Yoon Ju; Lee, Hong Je; Park, Ji Young; Jeong, Shin Young; Lee, Sang-Woo; Ha, Jeoung-Hee; Ahn, Byeong-Cheol; Jeon, Yong Hyun; Lee, Jaetae

    2015-01-01

    We sought to evaluate the feasibility of molecular imaging using the human sodium iodide symporter (hNIS) gene as a reporter, in addition to the enhanced firefly luciferase (effluc) gene, for tracking dendritic cell (DCs) migration in living mice. A murine dendritic cell line (DC2.4) co-expressing hNIS and effluc genes (DC/NF) was established. For the DC-tracking study, mice received either parental DCs or DC/NF cells in the left or right footpad, respectively, and combined I-124 PET/CT and bioluminescence imaging (BLI) were performed. In vivo PET/CT imaging with I-124 revealed higher activity of the radiotracer in the draining popliteal lymph nodes (DPLN) of the DC/NF injection site at day 1 than DC injection site (p < 0.05). The uptake value further increased at day 4 (p < 0.005). BLI also demonstrated migration of DC/NF cells to the DPLNs at day 1 post-injection, and signals at the DPLNs were much higher at day 4. These data support the feasibility of hNIS reporter gene imaging in the tracking of DC migration to lymphoid organs in living mice. DCs expressing the NIS reporter gene could be a useful tool to optimize various strategies of cell-based immunotherapy. PMID:25974752

  5. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  6. In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

    SciTech Connect

    Allegra, Danilo; Mertens, Daniel

    2011-03-25

    Research highlights: {yields} Posttranscriptional regulation of miRNA processing is difficult to quantify. {yields} Our in-vivo processing assay can quantify Drosha cleavage in live cells. {yields} It is based on luciferase reporters fused with pri-miRNAs. {yields} The assay validates the processing defect caused by a mutation in pri-16-1. {yields} It is a sensitive method to quantify pri-miRNA cleavage by Drosha in live cells. -- Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.

  7. Generation of Luciferase-Expressing Leishmania infantum chagasi and Assessment of Miltefosine Efficacy in Infected Hamsters through Bioimaging

    PubMed Central

    Reimão, Juliana Q.; Oliveira, Jordana C.; Trinconi, Cristiana T.; Cotrim, Paulo C.; Coelho, Adriano C.; Uliana, Silvia R. B.

    2015-01-01

    Background The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters. Methodology/Principal Findings A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival. Conclusions/Significance Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before

  8. Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

    PubMed Central

    2011-01-01

    Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi

  9. Improved luciferase gene expression using ultrasound targeted microbubble destruction therapy in swine

    NASA Astrophysics Data System (ADS)

    Noble, Misty L.; Song, Shuxian; Sun, Ryan R.; Fan, Luping; DiBlasi, Robert M.; O'Kelly-Priddy, Colleen; Loeb, Keith R.; Miao, Carol H.

    2012-11-01

    Ultrasound (US) targeted microbubble (MB) destruction (UTMD) has been shown to be an effective method in delivering drugs and plasmid DNA (pDNA) into cells. We previously reported successful gene transfection of a reporter luciferase gene, pGL4, into livers of mice and rats using UTMD. The challenge is to translate and achieve similar gene expression in large animals, like swine, where the treated tissue volume is substantially larger. The scale-up study requires proportionally increased amount of pDNA/MBs delivered to tissues and an equivalent increase in US energy. We use different MBs and surgical strategies to retain most of pDNA/MB locally during US application in order to maximize the effect of UTMD in gene transfection. Our results show significant increase in luciferase expression in swine injected with MBs and exposed to 2.7 MPa US. We obtained up to 1800-fold enhancement in the pig experiment using Definity® MBs, and 2000-fold and 6300-fold enhancement in two pig studies using RN18 MBs compared to sham. These results represent an important developmental step towards US mediated gene delivery in large animals and clinical trials.

  10. Technical planning activity: Final report

    SciTech Connect

    Not Available

    1987-01-01

    In April 1985, the US Department of Energy's (DOE's) Office of Fusion Energy commissioned the Technical Planning Activity (TPA). The purpose of this activity was to develop a technical planning methodology and prepare technical plans in support of the strategic and policy framework of the Magnetic Fusion Program Plan issued by DOE in February 1985. Although this report represents the views of only the US magnetic fusion community, it is international in scope in the sense that the technical plans contained herein describe the full scope of the tasks that are prerequisites for the commercialization of fusion energy. The TPA has developed a well-structured methodology that includes detailed definitions of technical issues, definitions of program areas and elements, statements of research and development objectives, identification of key decision points and milestones, and descriptions of facility requirements.

  11. A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

    SciTech Connect

    Shi, Junwei; Zhang, Huan; Fang, Liurong; Xi, Yongqiang; Zhou, Yanrong; Luo, Rui; Wang, Dang Xiao, Shaobo; Chen, Huanchun

    2014-10-03

    Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.

  12. Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ -ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation

    PubMed Central

    Jobe, Timothy O.; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2015-01-01

    Summary Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M2 seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione

  13. E-Division activities report

    SciTech Connect

    Barschall, H.H.

    1984-07-01

    E (Experimental Physics) Division carries out basic and applied research in atomic and nuclear physics, in materials science, and in other areas related to the missions of the Laboratory. Some of the activities are cooperative efforts with other divisions of the Laboratory, and, in a few cases, with other laboratories. Many of the experiments are directly applicable to problems in weapons and energy, some have only potential applied uses, and others are in pure physics. This report presents abstracts of papers published by E (Experimental Physics) Division staff members between July 1983 and June 1984. In addition, it lists the members of the scientific staff of the division, including visitors and students, and some of the assignments of staff members on scientific committees. A brief summary of the budget is included.

  14. Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases.

    PubMed

    Morita, Naoki; Haga, Sanae; Ohmiya, Yoshihiro; Ozaki, Michitaka

    2016-03-15

    We propose a new concept of tumor progression monitoring using dual luciferases in living animals to reduce stress for small animals and the cost of luciferin. The secreted Cypridina luciferase (CLuc) was used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase was used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Thus, the new monitoring systems that use dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals. PMID:26717897

  15. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    NASA Astrophysics Data System (ADS)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  16. A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

    PubMed

    Wang, Y; Wang, G; O'Kane, D J; Szalay, A A

    2001-01-01

    We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time. PMID:11212912

  17. Use of liposome-mediated DNA transfection to determine promoter activity in smooth muscle cells.

    PubMed

    Fabunmi, R P

    1999-01-01

    The transfer and expression of DNA plasmids containing promoter fragments of heterologous genes linked to reporter cDNAs in mammalian cells has become an invaluable technique for studying the regulation of gene expression. Several reporter genes such as luciferase, β-galactosidase, chloramphenicol acetyl transferase, and green flourescent protein are ideal to study promoter activities as their gene products are not endogenous to smooth muscle cells (SMC) and their expression can be readily detected using convenient assays (1). Among these genes, a popular choice is the firefly luciferase, as its expression can be easily detected in cells using a highly sensitive chemiluminescent assay (2). The firefly luciferase catalyses a rapid, ATP-dependent oxidation of the substrate, luciferin, which then emits light. Reactions catalyzed by firefly luciferase are: [Formula: see text]. PMID:21341022

  18. Full color modulation of firefly luciferase through engineering with unified Stark effect.

    PubMed

    Cai, Duanjun; Marques, Miguel A L; Nogueira, Fernando

    2013-11-01

    The firefly luciferase has been a unique marking tool used in various bioimaging techniques. Extensive color modulation is strongly required to meet special marking demands; however, intentional and accurate wavelength tuning has yet to be achieved. Here, we demonstrate that the color shift of the firefly chromophore (OxyLH2-1) by internal and external fields can be described as a unified Stark shift. Electrostatic microenvironmental effects on fluorescent spectroscopy are modeled in vacuo through effective electric fields by using time-dependent density functional theory. A complete visible fluorescence spectrum of firefly chromophore is depicted, which enables one to control the emission in a specific color. As an application, the widely observed pH-correlated color shift is proved to be associated with the local Stark field generated by the trace water-ions (vicinal hydronium and hydroxide ions) at active sites close to the OxyLH2-1. PMID:24087879

  19. Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.

    PubMed

    Poutiainen, Pekka K; Rönkkö, Teemu; Hinkkanen, Ari E; Palvimo, Jorma J; Närvänen, Ale; Turhanen, Petri; Laatikainen, Reino; Weisell, Janne; Pulkkinen, Juha T

    2014-01-15

    In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena. PMID:24341748

  20. A Cytoplasmic Form of Gaussia luciferase Provides a Highly Sensitive Test for Cytotoxicity

    PubMed Central

    Tsuji, Saori; Ohbayashi, Tetsuya; Yamakage, Kohji; Oshimura, Mitsuo; Tada, Masako

    2016-01-01

    The elimination of unfavorable chemicals from our environment and commercial products requires a sensitive and high-throughput in vitro assay system for drug-induced hepatotoxicity. Some previous methods for evaluating hepatotoxicity measure the amounts of cytoplasmic enzymes secreted from damaged cells into the peripheral blood or culture medium. However, most of these enzymes are proteolytically digested in the extracellular milieu, dramatically reducing the sensitivity and reliability of such assays. Other methods measure the decrease in cell viability following exposure to a compound, but such endpoint assays are often confounded by proliferation of surviving cells that replace dead or damaged cells. In this study, with the goal of preventing false-negative diagnoses, we developed a sensitive luminometric cytotoxicity test using a stable form of luciferase. Specifically, we converted Gaussia luciferase (G-Luc) from an actively secreted form to a cytoplasmic form by adding an ER-retention signal composed of the four amino acids KDEL. The bioluminescent signal was >30-fold higher in transgenic HepG2 human hepatoblastoma cells expressing G-Luc+KDEL than in cells expressing wild-type G-Luc. Moreover, G-Luc+KDEL secreted from damaged cells was stable in culture medium after 24 hr at 37°C. We evaluated the accuracy of our cytotoxicity test by subjecting identical samples obtained from chemically treated transgenic HepG2 cells to the G-Luc+KDEL assay and luminometric analyses based on secretion of endogenous adenylate kinase or cellular ATP level. Time-dependent accumulation of G-Luc+KDEL in the medium increased the sensitivity of our assay above those of existing tests. Our findings demonstrate that strong and stable luminescence of G-Luc+KDEL in human hepatocyte-like cells, which have high levels of metabolic activity, make it suitable for use in a high-throughput screening system for monitoring time-dependent cytotoxicity in a limited number of cells. PMID

  1. Multiplexing Bioluminescent and Fluorescent Reporters to Monitor Live Cells

    PubMed Central

    Haugwitz, Michael; Nourzaie, Omar; Garachtchenko, Tatiana; Hu, Lanrong; Gandlur, Suvarna; Olsen, Cathy; Farmer, Andrew; Chaga, Grigoriy; Sagawa, Hiroaki

    2008-01-01

    Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein. PMID:20161823

  2. Multiplexing bioluminescent and fluorescent reporters to monitor live cells.

    PubMed

    Haugwitz, Michael; Nourzaie, Omar; Garachtchenko, Tatiana; Hu, Lanrong; Gandlur, Suvarna; Olsen, Cathy; Farmer, Andrew; Chaga, Grigoriy; Sagawa, Hiroaki

    2008-01-01

    Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein. PMID:20161823

  3. Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems.

    PubMed

    Burbelo, Peter D; Ching, Kathryn H; Bren, Kathleen E; Iadarola, Michael J

    2011-06-01

    B-cell-mediated humoral responses are triggered in many human diseases, including autoimmune diseases, cancer, and neurologic and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed luciferase immunoprecipitation systems (LIPS) that harnesses light-emitting proteins to generate high-definition antibody profiles that are optimal for both diagnostics and biomarker discovery. Here, we describe the results and implications from a range of LIPS-antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis and offer a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring. PMID:21679112

  4. Active Matrix OLED Test Report

    NASA Technical Reports Server (NTRS)

    Salazar, George

    2013-01-01

    This report focuses on the limited environmental testing of the AMOLED display performed as an engineering evaluation by The NASA Johnson Space Center (JSC)-specifically. EMI. Thermal Vac, and radiation tests. The AMOLED display is an active-matrix Organic Light Emitting Diode (OLED) technology. The testing provided an initial understanding of the technology and its suitability for space applications. Relative to light emitting diode (LED) displays or liquid crystal displays (LCDs), AMOLED displays provide a superior viewing experience even though they are much lighter and smaller, produce higher contrast ratio and richer colors, and require less power to operate than LCDs. However, AMOLED technology has not been demonstrated in a space environment. Therefore, some risks with the technology must be addressed before they can be seriously considered for human spaceflight. The environmental tests provided preliminary performance data on the ability of the display technology to handle some of the simulated induced space/spacecraft environments that an AMOLED display will see during a spacecraft certification test program. This engineering evaluation is part of a Space Act Agreement (SM) between The NASA/JSC and Honeywell International (HI) as a collaborative effort to evaluate the potential use of AMOLED technology for future human spaceflight missions- both government-led and commercial. Under this SM, HI is responsible for doing optical performance evaluation, as well as temperature and touch screen studies. The NASA/JSC is responsible for performing environmental testing comprised of EMI, Thermal Vac, and radiation tests. Additionally, as part of the testing, limited optical data was acquired to assess performance as the display was subjected to the induced environments. The NASA will benefit from this engineering evaluation by understanding AMOLED suitability for future use in space as well as becoming a smarter buyer (or developer) of the technology. HI benefits

  5. Food flavonoid aryl hydrocarbon receptor-mediated agonistic/antagonistic/synergic activities in human and rat reporter gene assays.

    PubMed

    Van der Heiden, Edwige; Bechoux, Nathalie; Muller, Marc; Sergent, Thérèse; Schneider, Yves-Jacques; Larondelle, Yvan; Maghuin-Rogister, Guy; Scippo, Marie-Louise

    2009-04-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxin-responsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX((R)). Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cells were compared for species-dependent activities. We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24h. PMID:19286049

  6. Proposed ionic bond between Arg300 and Glu270 and Glu271 are not involved in inactivation of a mutant firefly luciferase (LRR).

    PubMed

    Sobhani-Damavandifar, Zahra; Hosseinkhani, Saman; Sajedi, Reza H

    2016-05-01

    The weakness of firefly luciferase is its rapid inactivation. Many studies have been done to develop thermostable luciferases. One of these modifications was LRR mutant in which the Leu300 was substituted with Arg in the E(354)RR(356)Lampyris turkestanicus luciferase as template. LRR was more thermostable than the wild type but with only 0.02% activity. In this study, site-directed mutagenesis was used to change the proposed ionic bond between the Arg and two neighboring residues (Glu270 and Glu271), to understand if the induced interactions were responsible for inactivation in LRR. Our results showed that substitution of Glu270 and 271 with Ala removed the interactions but the activity of enzyme did not return. The E270A mutant was more active than LRR but the E271A and E270A/E271A mutants were inactive. Fluorescence and CD measurements showed that these mutations were accompanied by conformational changes. Extrinsic fluorescence measurement and obtained quenching data by KI and acrylamide also confirmed that the mutants were less compact than the LRR enzyme. In conclusion, in LRR, the interactions between Arg300 and Glu270 and Glu271 were not responsible for the enzyme inactivation and it is proposed that the enzyme inactivation is due to conformational changes of LRR mutant of firefly luciferase. PMID:26992788

  7. Daily Physical Activity Survey Report

    ERIC Educational Resources Information Center

    Alberta Education, 2008

    2008-01-01

    The intent of the Daily Physical Activity (DPA) Survey was to gather school-level information from teachers and principals regarding their perceptions of DPA, thus providing a greater understanding of DPA implementation in grades 1 to 9. This study aimed to help identify the many variables that influence the attainment of the DPA outcomes and…

  8. Analysis of LPS-induced, NFκB-dependent interleukin-8 transcription in kidney embryonic cell line expressing TLR4 using luciferase assay.

    PubMed

    Yunusova, Tamara; Akhtar, Mumtaz; Poltoratsky, Vladimir

    2014-01-01

    Gene expression is orchestrated by a complex network of signal transduction pathways that typically originate on cell surface receptors and culminate in DNA-binding transcription factors, which translocate to the nucleus and bind cis-regulatory elements in promoter regions of genes, thereby inducing de novo synthesis of the nascent RNA transcripts and their splicing. Gene expression arrays monitor abundance of the matured, spliced cDNA, which undergoes additional posttranscriptional modifications that greatly affect the half-life of the cDNA. Thus, the relative abundance of cDNA is not necessarily commensurable with the activity of promoters of the corresponding genes. In contrast, reporter gene assays provide valuable insight into the regulation of gene expression at the level of transcription and allow for discerning the contribution of individual transcription factors into changes in gene expression. Here, we describe a robust reporter gene assay method that is useful for exploration of transcription regulatory network, which regulates gene expression in response to inflammation. The method is exemplified by using the promoter region of the prototypic pro-inflammatory chemokine interleukin-8 (IL-8, CXCL8), which plays an important role in immune response as well as carcinogenesis. Using the luciferase reporter gene assay, we analyze the activation status of the IL-8 promoter in lipopolysaccharide (LPS)-stimulated human embryonic kidney cells. PMID:24908317

  9. Activities report in applied physics

    NASA Astrophysics Data System (ADS)

    Research concerning acoustics, heat, architecture, materials research, and (optical) instrumentation is presented; active noise control and acoustic path identification were investigated. Energy conservation, solar energy, and building physics activities were carried out. Ultraviolet absorbing glasses, glass fibers, sheet glass, and aluminium and silicon oxynitrides, were studied. Glass fiber based sensor and laser applications, and optical space-instrumentation are discussed. Signal processing, sensors, and integrated electronics applications were developed. Scale model experiments for flow induced noise and vibrations, caused by engines, ventilators, wind turbines, and propellers, were executed. A multispectral charge coupled device airborne scanner, with four modules (one for forward observations) is described. A ground radar, based on seismic exploration signal processing and used for the location of pipes, sewers and cables, was developed.

  10. Activities report of PTT Research

    NASA Astrophysics Data System (ADS)

    In the field of postal infrastructure research, activities were performed on postcode readers, radiolabels, and techniques of operations research and artificial intelligence. In the field of telecommunication, transportation, and information, research was made on multipurpose coding schemes, speech recognition, hypertext, a multimedia information server, security of electronic data interchange, document retrieval, improvement of the quality of user interfaces, domotics living support (techniques), and standardization of telecommunication prototcols. In the field of telecommunication infrastructure and provisions research, activities were performed on universal personal telecommunications, advanced broadband network technologies, coherent techniques, measurement of audio quality, near field facilities, local beam communication, local area networks, network security, coupling of broadband and narrowband integrated services digital networks, digital mapping, and standardization of protocols.

  11. Apollo experience report: Safety activities

    NASA Technical Reports Server (NTRS)

    Rice, C. N.

    1975-01-01

    A description is given of the flight safety experiences gained during the Apollo Program and safety, from the viewpoint of program management, engineering, mission planning, and ground test operations was discussed. Emphasis is placed on the methods used to identify the risks involved in flight and in certain ground test operations. In addition, there are discussions on the management and engineering activities used to eliminate or reduce these risks.

  12. An improved thyroid hormone reporter assay to determine the thyroid hormone-like activity of amiodarone, bithionol, closantel and rafoxanide.

    PubMed

    Matsubara, Kana; Sanoh, Seigo; Ohta, Shigeru; Kitamura, Shigeyuki; Sugihara, Kazumi; Fujimoto, Nariaki

    2012-01-01

    A number of environmental chemicals have been reported to exhibit thyroid hormone-like activity. Since thyroid hormones play a crucial role in development, it is important to identify chemicals in the environment that are capable of endocrine disruption of thyroid hormone homeostasis. In order to detect thyroid hormone-like activity, the growth of pituitary cell lines has been commonly used as a sensitive marker, albeit with limited specificity to thyroid hormones. Reporter gene assays using the thyroid hormone responsive element (TRE) connected to the luciferase reporter gene have also been developed. Thus far however, this type of assay appears to have limited sensitivity compared to cell growth assays. In the present study, we developed a highly sensitive TRE reporter gene assay by using a pituitary cell line, MtT/E-2, and by culturing cells in a serum-free medium. Our assay was developed in order to detect T3 activity at a concentration of 10(-11)M. This assay identified thyroid hormone-like activity from the antiarrhythmic drug, amiodarone, and from three anti-parasitic drugs, bithionol, closantel and rafoxanide, all commonly used in veterinary medicine. Thyroid hormone-like activity of these compounds was further confirmed by the induction of BCL3 gene expression in MtT/E-2, which is known to be regulated by thyroid hormones. Our improved assay was proved to be a sensitive tool for assessing thyroid hormone-like activity of environmental chemicals. PMID:22015988

  13. FIREFLY LUCIFERASE ATP ASSAY DEVELOPMENT FOR MONITORING BACTERIAL CONCENTRATIONS IN WATER SUPPLIES

    EPA Science Inventory

    This research program was initiated to develop a rapid, automatable system for measuring total viable microorganisms in potable drinking water supplies using the firefly luciferase ATP assay. The assay was adapted to an automatable flow system that provided comparable sensitivity...

  14. A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases

    PubMed Central

    Jester, Benjamin W.; Cox, Kurt J.; Gaj, Alicia; Shomin, Carolyn D.; Porter, Jason R.; Ghosh, Indraneel

    2010-01-01

    The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors. PMID:20669947

  15. Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system.

    PubMed

    Lwa, Teng Rhui; Tan, Chuan Hao; Lew, Qiao Jing; Chu, Kai Ling; Tan, Janice; Lee, Yih Yean; Chao, Sheng-Hao

    2010-04-15

    Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells. PMID:20188772

  16. Advanced Light Source Activity Report 2000

    SciTech Connect

    Greiner, A.; Moxon, L.; Robinson, A.; Tamura, L.

    2001-04-01

    This is an annual report, detailing activities at the Advanced Light Source for the year 2000. It includes highlights of scientific research by users of the facility as well as information about the development of the facility itself.

  17. Report on observational activity in (summer) 2015

    NASA Astrophysics Data System (ADS)

    Zejda, M.

    2016-03-01

    A short report on the author's observational activity in 2015 and the last 20 years is given. In total this means almost 900 nights, about a half million of CCD frames and thousands of photometric measurements.

  18. Reporters to monitor cellular MMP12 activity

    NASA Astrophysics Data System (ADS)

    Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

    2010-02-01

    Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

  19. Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation.

    PubMed

    Fortin, Pierre-Yves; Genevois, Coralie; Chapolard, Mathilde; Santalucía, Tomàs; Planas, Anna M; Couillaud, Franck

    2014-02-01

    Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins. A double transgenic mouse strain expressing the firefly luciferase (lucF) and fluorescent mPlum protein under the transcriptional control of the thermo-inducible heat-shock protein (Hspa1b) promoter was generated allowing to follow up the reporter proteins by different and complementary in vivo imaging technologies. These mice were used for in vivo imaging by bioluminescence and epi fluorescence reflectance imaging (BLI & FRI) and as a source of embryonic fibroblast (MEF) for in vitro approaches. LucF, mPlum and endogenous Hsp70 mRNAs were transcribed simultaneously. The increase in mRNA was transient, peaking at 3 h and then returning to the basal level about 6 h after the thermal stimulations. The bioluminescent signal was transient and initiated with a 3 h delay versus mRNA expression. The onset of mPlum fluorescence was more delayed, increasing slowly up to 30 h after heat-shock and remaining for several days. This mouse allows for both bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) of Hsp70 promoter activation showing an early and transient lucF activity and a retrospective and persistent mPlum fluorescence. This transgenic mouse will allow following the transient local induction of Hsp-70 promoter beyond its induction time-frame and relate into subsequent dynamic biological effects of the heat-shock response. PMID:24575340

  20. Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation

    PubMed Central

    Fortin, Pierre-Yves; Genevois, Coralie; Chapolard, Mathilde; Santalucía, Tomàs; Planas, Anna M.; Couillaud, Franck

    2014-01-01

    Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins. A double transgenic mouse strain expressing the firefly luciferase (lucF) and fluorescent mPlum protein under the transcriptional control of the thermo-inducible heat-shock protein (Hspa1b) promoter was generated allowing to follow up the reporter proteins by different and complementary in vivo imaging technologies. These mice were used for in vivo imaging by bioluminescence and epi fluorescence reflectance imaging (BLI & FRI) and as a source of embryonic fibroblast (MEF) for in vitro approaches. LucF, mPlum and endogenous Hsp70 mRNAs were transcribed simultaneously. The increase in mRNA was transient, peaking at 3 h and then returning to the basal level about 6 h after the thermal stimulations. The bioluminescent signal was transient and initiated with a 3 h delay versus mRNA expression. The onset of mPlum fluorescence was more delayed, increasing slowly up to 30 h after heat-shock and remaining for several days. This mouse allows for both bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) of Hsp70 promoter activation showing an early and transient lucF activity and a retrospective and persistent mPlum fluorescence. This transgenic mouse will allow following the transient local induction of Hsp-70 promoter beyond its induction time-frame and relate into subsequent dynamic biological effects of the heat-shock response. PMID:24575340

  1. Status report of RMS active damping augmentation

    NASA Technical Reports Server (NTRS)

    Gilbert, Mike; Demeo, Martha E.

    1993-01-01

    A status report of Remote Manipulator System (RMS) active damping augmentation is presented. Topics covered include: active damping augmentation; benefits of RMS ADA; simulated payload definition; sensor and actuator definition; ADA control law design; Shuttle Engineering Simulator (SES) real-time simulation; and astronaut evaluation.

  2. Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

    PubMed Central

    Krishnamoorthy, Archana

    2015-01-01

    Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases. PMID:26162874

  3. Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays.

    PubMed

    Legler, Juliette; Dennekamp, Martine; Vethaak, A Dick; Brouwer, Abraham; Koeman, Jan H; van der Burg, Bart; Murk, Albertinka J

    2002-07-01

    Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations. PMID:12109482

  4. Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery

    PubMed Central

    Che, Pulin; Cui, Long; Kutsch, Olaf; Cui, Liwang

    2012-01-01

    Abstract The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a high-throughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z′-value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC1280 small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z′-value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery. PMID:22050430

  5. Self-Assembling NanoLuc Luciferase Fragments as Probes for Protein Aggregation in Living Cells.

    PubMed

    Zhao, Jia; Nelson, Travis J; Vu, Quyen; Truong, Tiffany; Stains, Cliff I

    2016-01-15

    Given the clear role of protein aggregation in human disease, there is a critical need for assays capable of quantifying protein aggregation in living systems. We hypothesized that the inherently low background and biocompatibility of luminescence signal readouts could provide a potential solution to this problem. Herein, we describe a set of self-assembling NanoLuc luciferase (Nluc) fragments that produce a tunable luminescence readout that is dependent upon the solubility of a target protein fused to the N-terminal Nluc fragment. To demonstrate this approach, we employed this assay in bacteria to assess mutations known to disrupt amyloid-beta (Aβ) aggregation as well as disease-relevant mutations associated with familial Alzheimer's diseases. The luminescence signal from these experiments correlates with the reported aggregation potential of these Aβ mutants and reinforces the increased aggregation potential of disease-relevant mutations in Aβ1-42. To further demonstrate the utility of this approach, we show that the effect of small molecule inhibitors on Aβ aggregation can be monitored using this system. In addition, we demonstrate that aggregation assays can be ported into mammalian cells. Taken together, these results indicate that this platform could be used to rapidly screen for mutations that influence protein aggregation as well as inhibitors of protein aggregation. This method offers a novel, genetically encodable luminescence readout of protein aggregation in living cells. PMID:26492083

  6. Establishment of ATP-based luciferase viability assay in 96-well plate for Trypanosoma congolense.

    PubMed

    Suganuma, Keisuke; Allamanda, Puttik; Hakimi, Hassan; Zhou, Mo; Angeles, Jose Ma; Kawazu, Shin-ichiro; Inoue, Noboru

    2014-11-01

    Animal African trypanosomosis (AAT), caused by Trypanosoma congolense, is widespread throughout sub-Saharan Africa. There are significant concerns related to the current drugs available for the treatment of AAT due to their limited effectiveness across species and their adverse effects. Moreover, drug resistant trypanosomes have recently been reported in the field. High throughput screening (HTS) of large chemical compound library collections is a promising approach for identifying novel drug candidates. While HTS for Trypanozoon trypanosomes, T. brucei sspp. and T. evansi is well established, no assays have been developed for T. congolense. In the present study, the authors developed an ATP-based luciferase viability assay for T. congolense in a 96-well plate format. The calculated 50% inhibitory concentration (IC50) values for pentamidine and diminazene were 10-100 times higher in T. congolense than in T. brucei. This result suggests that the transporters for the 2 tested compounds differ between T. congolense and T. brucei. This assay could further be applied to screen novel chemical compounds for the treatment of AAT caused by T. congolense. PMID:25056575

  7. Establishment of ATP-Based Luciferase Viability Assay in 96-Well Plate for Trypanosoma congolense

    PubMed Central

    SUGANUMA, Keisuke; ALLAMANDA, Puttik; HAKIMI, Hassan; ZHOU, Mo; ANGELES, Jose Ma.; KAWAZU, Shin-ichiro; INOUE, Noboru

    2014-01-01

    ABSTRACT Animal African trypanosomosis (AAT), caused by Trypanosoma congolense, is widespread throughout sub-Saharan Africa. There are significant concerns related to the current drugs available for the treatment of AAT due to their limited effectiveness across species and their adverse effects. Moreover, drug resistant trypanosomes have recently been reported in the field. High throughput screening (HTS) of large chemical compound library collections is a promising approach for identifying novel drug candidates. While HTS for Trypanozoon trypanosomes, T. brucei sspp. and T. evansi is well established, no assays have been developed for T. congolense. In the present study, the authors developed an ATP-based luciferase viability assay for T. congolense in a 96-well plate format. The calculated 50% inhibitory concentration (IC50) values for pentamidine and diminazene were 10–100 times higher in T. congolense than in T. brucei. This result suggests that the transporters for the 2 tested compounds differ between T. congolense and T. brucei. This assay could further be applied to screen novel chemical compounds for the treatment of AAT caused by T. congolense. PMID:25056575

  8. Probing Bioluminescence Resonance Energy Transfer in Quantum Rod-Luciferase Nanoconjugates.

    PubMed

    Alam, Rabeka; Karam, Liliana M; Doane, Tennyson L; Coopersmith, Kaitlin; Fontaine, Danielle M; Branchini, Bruce R; Maye, Mathew M

    2016-02-23

    We describe the necessary design criteria to create highly efficient energy transfer conjugates containing luciferase enzymes derived from Photinus pyralis (Ppy) and semiconductor quantum rods (QRs) with rod-in-rod (r/r) microstructure. By fine-tuning the synthetic conditions, CdSe/CdS r/r-QRs were prepared with two different emission colors and three different aspect ratios (l/w) each. These were hybridized with blue, green, and red emitting Ppy, leading to a number of new BRET nanoconjugates. Measurements of the emission BRET ratio (BR) indicate that the resulting energy transfer is highly dependent on QR energy accepting properties, which include absorption, quantum yield, and optical anisotropy, as well as its morphological and topological properties, such as aspect ratio and defect concentration. The highest BR was found using r/r-QRs with lower l/w that were conjugated with red Ppy, which may be activating one of the anisotropic CdSe core energy levels. The role QR surface defects play on Ppy binding, and energy transfer was studied by growth of gold nanoparticles at the defects, which indicated that each QR set has different sites. The Ppy binding at those sites is suggested by the observed BRET red-shift as a function of Ppy-to-QR loading (L), where the lowest L results in highest efficiency and furthest shift. PMID:26760436

  9. Recombinant receptor/reporter gene bioassays for assessing the estrogenic and dioxin-like activities of xenobiotics and complex mixtures

    SciTech Connect

    Zacharewski, T.

    1995-12-31

    Exposure to naturally occurring or synthetic substances that possess sex steroid and/or dioxin-like activity may have long range effects on human health, reproductive fitness and environmental quality. Results from recent epidemiological studies have suggested that xenobiotics with sex steroid activity may contribute to the development of hormone-dependent cancers and disorders in the male reproductive tract as well as attenuate sperm production. However, most of these compounds, which are referred to as endocrine disruptors, are structurally dissimilar to sex steroids. Yet, based upon ambiguous assays, it has been conceded that the effects of these compounds are mediated by receptors. The authors have taken advantage of the mechanism of action of these compounds to develop recombinant receptor/reporter gene bioassays for environmental estrogens and dioxin-like compounds. The assays use an easily measurable enzyme activity (i.e. firefly luciferase), exhibit improved sensitivity and selectivity and are amenable to automation. Data will be presented demonstrating that phytoestrogens (e.g. genistein) and xenobiotics such as pesticides (e.g. DDT, Kepone), nonionic surfactants (e.g. p-nonylphenol), and precursors used in the manufacture of plastics (e.g. Bisphenol A) exhibit estrogenic activity. In addition, the assays have been used to detect estrogenic and dioxin-like activity in complex mixtures such as pulp and paper mill black liquor and effluent. These results demonstrate the utility of recombinant receptor/reporter gene bioassays for identifying substances or complex mixtures with estrogenic and/or dioxin-like activity.

  10. Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

    SciTech Connect

    Cruz, P.G.; Auld, D.S.; Schultz, P.J.; Lovell, S.; Battaile, K.P.; MacArthur, R.; Shen, M.; Tamayo-Castillo, G.; Inglese, J.; Sherman, D.H.

    2011-11-28

    The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves obtained from quantitative HTS to improve the initial selection of actives from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm improves the probability that labor-intensive subsequent steps of reculturing, extraction, and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by X-ray crystallography.

  11. Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

    PubMed Central

    Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

    2010-01-01

    Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

  12. Firefly Luciferase-Based Sequential Bioluminescence Resonance Energy Transfer (BRET)-Fluorescence Resonance Energy Transfer (FRET) Protease Assays.

    PubMed

    Branchini, Bruce

    2016-01-01

    We describe here the preparation of ratiometric luminescent probes that contain two well-separated emission peaks produced by a sequential bioluminescence resonance energy transfer (BRET)-fluorescence resonance energy transfer (FRET) process. The probes are single soluble fusion proteins consisting of a thermostable firefly luciferase variant that catalyzes yellow-green (560 nm maximum) bioluminescence and a red fluorescent protein covalently labeled with a near-Infrared fluorescent dye. The two proteins are connected by a decapeptide containing a protease recognition site specific for factor Xa, thrombin, or caspase 3. The rates of protease cleavage of the fusion protein substrates were monitored by recording emission spectra and plotting the change in peak ratios over time. Detection limits of 0.41 nM for caspase 3, 1.0 nM for thrombin, and 58 nM for factor Xa were realized with a scanning fluorometer. This method successfully employs an efficient sequential BRET-FRET energy transfer process based on firefly luciferase bioluminescence to assay physiologically important protease activities and should be generally applicable to the measurement of any endoprotease lacking accessible cysteine residues. PMID:27424898

  13. [Photoreactivation of UV-irradiated Escherichia coli K12 AB1886 uvrA6 with assistance of luminescence of Photobacterium leiognathi Luciferase].

    PubMed

    Melkina, O E; Kotova, V Yu; Konopleva, M N; Manukhov, I V; Pustovoit, K S; Zavilgelsky, G B

    2015-01-01

    The bioluminescence induced by luciferases of marine bacteria promotes repair of UV damaged DNA of Escherichia coli AB1886 uvrA6. It is shown that bacterial photolyase that implements photoreactivation activity is the major contributor to DNA repair. However, the intensity of bioluminescence increasing induced by UV-irradiation (SOS-induction) in bacterial cells is not enough for efficient photoreactivation. PMID:26710787

  14. Total Synthesis and Biological Studies of TMC-205 and Analogues as Anticancer Agents and Activators of SV40 Promoter

    PubMed Central

    2014-01-01

    TMC-205 is a natural fungal metabolite with antiproliferative activity against cancer cell lines. The light- and air-sensitivity prevented in-depth exploitation of this novel indole derivative. Herein, we report the first synthesis of TMC-205. On the basis of its reactivity with reactive oxygen species, we developed air-stable analogues of TMC-205. These analogues are 2–8-fold more cytotoxic than TMC-205 against HCT-116 colon cancer cell line. Importantly, at noncytotoxic dose levels, these analogues activated the transcription of luciferase reporter gene driven by simian virus 40 promoter (SV40). Further, these small molecules also inhibit firefly luciferase, presumably by direct interaction. PMID:25147604

  15. Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

    PubMed

    Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

    2016-01-01

    The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

  16. Light without substrate amendment: the bacterial luciferase gene cassette as a mammalian bioreporter

    NASA Astrophysics Data System (ADS)

    Close, Dan M.; Xu, Tingting; Smartt, Abby E.; Jegier, Pat; Ripp, Steven A.; Sayler, Gary S.

    2011-06-01

    Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor development.

  17. Establishment of a novel method to evaluate peritoneal microdissemination and therapeutic effect using luciferase assay.

    PubMed

    Takahashi, Ryo; Yokobori, Takehiko; Osone, Katsuya; Tatsuki, Hironori; Takada, Takahiro; Suto, Toshinaga; Yajima, Reina; Kato, Toshihide; Fujii, Takaaki; Tsutsumi, Souichi; Kuwano, Hiroyuki; Asao, Takayuki

    2016-03-01

    Peritoneal dissemination is a major cause of recurrence in patients with malignant tumors in the peritoneal cavity. Effective anticancer agents and treatment protocols are necessary to improve outcomes in these patients. However, previous studies using mouse models of peritoneal dissemination have not detected any drug effect against peritoneal micrometastasis. Here we used the luciferase assay to evaluate peritoneal micrometastasis in living animals and established an accurate mouse model of early peritoneal microdissemination to evaluate tumorigenesis and drug efficacy. There was a positive correlation between luminescence intensity in in vivo luciferase assay and the extent of tumor dissemination evaluated by ex vivo luciferase assay and mesenteric weight. This model has advantages over previous models because optimal luciferin concentration without cell damage was validated and peritoneal microdissemination could be quantitatively evaluated. Therefore, it is a useful model to validate peritoneal micrometastasis formation and to evaluate drug efficacy without killing mice. PMID:26716425

  18. Problem areas in the use of the firefly luciferase assay for bacterial detection

    NASA Technical Reports Server (NTRS)

    Picciolo, G. L.; Chappelle, E. W.; Knust, E. A.; Tuttle, S. A.; Curtis, C. A.

    1975-01-01

    By purifying the firefly luciferase extract and adding all necessary chemicals but ATP in excess, an assay for ATP was performed by measuring the amount of light produced when a sample containing soluble ATP is added to the luciferase reaction mixture. Instrumentation, applications, and basic characteristics of the luciferase assay are presented. Effect of the growth medium and length of time grown in this medium on ATP per viable E. coli values is shown in graphic form, along with an ATP concentration curve showing relative light units versus ATP injected. Reagent functions and concentration methods are explored. Efforts to develop a fast automatable system to detect the presence of bacteria in biological fluids, especially urine, resulted in the optimization of procedures for use with different types of samples.

  19. "In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

    ERIC Educational Resources Information Center

    Higgins, Pamela J.

    2005-01-01

    This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

  20. Physics Division activities report, 1986--1987

    SciTech Connect

    Not Available

    1987-01-01

    This report summarizes the research activities of the Physics Division for the years 1986 and 1987. Areas of research discussed in this paper are: research on e/sup +/e/sup /minus// interactions; research on p/bar p/ interactions; experiment at TRIUMF; double beta decay; high energy astrophysics; interdisciplinary research; and advanced technology development and the SSC.

  1. Annual waste reduction activities report. Issue 1

    SciTech Connect

    1991-03-18

    This report discusses the waste minimization activities for the Pinellas Plant. The Pinellas Plant deals with low-level radioactive wastes, solvents, scrap metals and various other hazardous materials. This program has realized cost savings through recycling and reuse of materials.

  2. Rational design of a triple reporter gene for multimodality molecular imaging.

    PubMed

    Hsieh, Ya-Ju; Hwu, Luen; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Wang, Hsin-Ell; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

    2014-01-01

    Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

  3. Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

    PubMed

    Ajit, Deepa; Simonyi, Agnes; Li, Runting; Chen, Zihong; Hannink, Mark; Fritsche, Kevin L; Mossine, Valeri V; Smith, Robert E; Dobbs, Thomas K; Luo, Rensheng; Folk, William R; Gu, Zezong; Lubahn, Dennis B; Weisman, Gary A; Sun, Grace Y

    2016-07-01

    The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells. PMID:27166148

  4. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    SciTech Connect

    Manning, Gillian E.; Mundy, Lukas J.; Crump, Doug; Jones, Stephanie P.; Chiu, Suzanne; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of toxicity than CYP

  5. Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

    PubMed

    Viviani, Vadim R; Neves, Deimison Rodrigues; Amaral, Danilo Trabuco; Prado, Rogilene A; Matsuhashi, Takuto; Hirano, Takashi

    2014-08-19

    Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar

  6. Aerosol delivery of kinase-deficient Akt1 attenuates Clara cell injury induced by naphthalene in the lungs of dual luciferase mice.

    PubMed

    Minai-Tehrani, Arash; Park, Young-Chan; Hwang, Soon-Kyung; Kwon, Jung-Taek; Chang, Seung-Hee; Park, Sung-Jin; Yu, Kyeong-Nam; Kim, Ji-Eun; Shin, Ji-Young; Kim, Ji-Hye; Kang, Bitna; Hong, Seong-Ho; Cho, Myung-Haing

    2011-12-01

    Conventional lung cancer therapies are associated with poor survival rates; therefore, new approaches such as gene therapy are required for treating cancer. Gene therapies for treating lung cancer patients can involve several approaches. Among these, aerosol gene delivery is a potentially more effective approach. In this study, Akt1 kinase-deficient (KD) and wild-type (WT) Akt1 were delivered to the lungs of CMV-LucR-cMyc-IRES-LucF dual reporter mice through a nose only inhalation system using glucosylated polyethylenimine and naphthalene was administrated to the mice via intraperitoneal injection. Aerosol delivery of Akt1 WT and naphthalene treatment increased protein levels of downstream substrates of Akt signaling pathway while aerosol delivery of Akt1 KD did not. Our results showed that naphthalene affected extracellular signal-regulated kinase (ERK) protein levels, ERK-related signaling, and induced Clara cell injury. However, Clara cell injury induced by naphthalene was considerably attenuated in mice exposed to Akt1 KD. Furthermore, a dual luciferase activity assay showed that aerosol delivery of Akt1 WT and naphthalene treatment enhanced cap-dependent protein translation, while reduced cap-dependent protein translation was observed after delivering Akt1 KD. These studies demonstrated that our aerosol delivery is compatible for in vivo gene delivery. PMID:22122896

  7. RNA interference screening of interferon-stimulated genes with antiviral activities against classical swine fever virus using a reporter virus.

    PubMed

    Wang, Xiao; Li, Yongfeng; Li, Lian-Feng; Shen, Liang; Zhang, Lingkai; Yu, Jiahui; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2016-04-01

    Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is a highly contagious and often fatal disease of pigs, which leads to significant economic losses in many countries. Viral infection can induce the production of interferons (IFNs), giving rise to the transcription of hundreds of IFN-stimulated genes (ISGs) to exert antiviral effects. Although numerous ISGs have been identified to possess antiviral activities against different viruses, rare anti-CSFV ISGs have been reported to date. In this study, to screen anti-CSFV ISGs, twenty-one ISGs reported previously were individually knocked down using small interfering RNAs (siRNAs) followed by infection with a reporter CSFV expressing Renilla luciferase (Rluc). As a result, four novel anti-CSFV ISGs were identified, including natural-resistance-associated macrophage protein 1 (NRAMP1), cytosolic 5'-nucleotidase III A (NT5C3A), chemokine C-X-C motif ligand 10 (CXCL10), and 2'-5'-oligoadenylate synthetase 1 (OAS1), which were further verified to exhibit antiviral activities against wild-type CSFV. We conclude that the reporter virus is a useful tool for efficient screening anti-CSFV ISGs. PMID:26868874

  8. Protein sterilization method of firefly luciferase using reduced pressure and molecular sieves

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Rich, E., Jr. (Inventor)

    1973-01-01

    The sterilization of the protein fruitfly luciferase under conditions that prevent denaturation is examined. Denaturation is prevented by heating the protein in contact with molecular seives and under a reduced pressure of the order of 0.00005 millimeters of mercury.

  9. Plasmodium falciparum Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors

    PubMed Central

    Elsworth, Brendan; Charnaud, Sarah C.; Sanders, Paul R.; Crabb, Brendan S.; Gilson, Paul R.

    2014-01-01

    Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients. PMID:25392998

  10. Plasmodium falciparum transfected with ultra bright NanoLuc luciferase offers high sensitivity detection for the screening of growth and cellular trafficking inhibitors.

    PubMed

    Azevedo, Mauro F; Nie, Catherine Q; Elsworth, Brendan; Charnaud, Sarah C; Sanders, Paul R; Crabb, Brendan S; Gilson, Paul R

    2014-01-01

    Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in Plasmodium falciparum and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients. PMID:25392998

  11. F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

    SciTech Connect

    Wu, Anna M

    2013-01-18

    The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

  12. The Bioluminescence Resonance Energy Transfer from Firefly Luciferase to a Synthetic Dye and its Application for the Rapid Homogeneous Immunoassay of Progesterone.

    PubMed

    Smirnova, Daria V; Samsonova, Jeanne V; Ugarova, Natalia N

    2016-01-01

    The sensitive BRET system for the homogeneous immunoassay of a low-molecular weight antigen was developed using progesterone as an example. Two thermostable mutants of the Luciola mingrelica firefly luciferase (Luc)-the "red" mutant with λmax.em = 590 nm (RedLuc) and the "green" mutant with λmax.em = 550 nm (GreenLuc)-were tested as the donors. The water-soluble Alexa Fluor 610× (AF) dye was selected as the acceptor because its two absorption maxima, located at 550 and 610 nm, are close to the bioluminescence maxima of the GreenLuc and RedLuc, respectively. The methods for the synthesis of the luciferase-progesterone (Luc-Pg) conjugate and the conjugate of the dye and the polyclonal antiprogesterone antibody (AF-Ab) were developed. Both conjugates retained their functional properties, had high antigen-antibody binding activity, and demonstrated a high BRET signal. The homogeneous immunoassay system based on the BRET from the firefly luciferase to the synthetic dye was established to assay progesterone as a model antigen. Optimization of the assay conditions, the composition of the reaction mixture, and the concentrations of the donor and the acceptor made it possible to reach the minimum detectable progesterone concentration of 0.5 ng mL(-1) . PMID:26650341

  13. Photodynamic therapy using luciferase nanoconjugate as a treatment for colon cancer

    NASA Astrophysics Data System (ADS)

    Koritarov, Tamara

    Photodynamic Therapy (PDT) has proven itself in previous studies to be a successful therapeutic treatment for surface tumors, but its effectiveness is limited to only shallow depths that allow for the penetration of light. This study demonstrates that we have improved upon the conventional method of PDT and have overcome the previous depth limitation by creating the light at the location of the tumor in situ. We conjugated a bioluminescent protein, Luciferase, to a semiconductor nanoparticle, TiO2, and with a cell specific antibody, anti-EGFR monoclonal antibody C225. The nanoconjugate, TiDoL-C225, was then activated by ATP and Luciferin in a reaction that creates reactive oxygen species (ROS) and induces apoptosis in the tumor cells. We created the optimal nanoconjugate synthesis protocol to make TiDoL and TiDoL-C225 for use in the PDT treatment. The TiDoL-C225 nanoconjugate is able to bind specifically to colon caner cells as the C225 antibody recognizes EGFR expressed at the surface of the cells, and further, when activated it will react only with the tumor cells. The optimal cell staining protocols were developed to visualize the treatment process and later analyze with the laser confocal microscope. The TiDoL nanoconjugate was found to only be operational and effective at killing tumor cells after being activated by Luciferin and ATP, which then enhances the control we have over the therapy. The TiDoL-C225 nanoconjugate increases the efficacy of binding to tumor cells and the speed of the reaction in the cells to begin apoptosis, even in lower concentrations when compared to the free TiDoL nanoconjugate. Finally, our PDT technique allowed us to monitor the tumor cells as they begin to undergo apoptosis in less than five minutes after the Luciferin was added to activate the reaction. The advantage of our method of PDT with the TiDoL-C225 nanoconjugate is that it can be used for early detection as well as developed into an effective treatment for cancers in all

  14. DETECTION OF ANDROGENIC ACTIVITY IN EMISSIONS FROM DIESEL FUEL AND BIOMASS COMBUSTION

    EPA Science Inventory

    The present study evaluated both diesel fuel exhaust and biomass (wood) burn extracts for androgen receptor¿mediated activity using MDA-kb2 cells, which contain an androgen-responsive promoter-luciferase reporter gene construct. This assay and analytical fractionization of the sa...

  15. Advanced light source. Activity report 1995

    SciTech Connect

    1996-07-01

    The ALS Activity Report is designed to share the breadth, variety, and interest of the scientific program and ongoing R&D efforts in a form that is accessible to a broad audience. Recent research results are presented in six sections, each representing an important theme in ALS science. These results are designed to demonstrate the capabilities of the ALS, rather than to give a comprehensive review of 1995 experiments. Although the scientific program and facilities report are separate sections, in practice the achievements and accomplishments of users and ALS staff are interdependent. This user-staff collaboration is essential to help us direct our efforts toward meeting the needs of the user community, and to ensure the continued success of the ALS as a premier facility.

  16. Cellular Bioenergetics is an Important Determinant of the Molecular Imaging Signal Derived from Luciferase and the Sodium-Iodide Symporter

    PubMed Central

    Chang, Connie; Chan, Angel; Lin, Xiaoping; Higuchi, Takahiro; Terrovitis, John; Afzal, Junaid M.; Rittenbach, Andrew; Sun, Dongdong; Vakrou, Styliani; Woldemichael, Kirubel; O’Rourke, Brian; Wahl, Richard; Pomper, Martin; Tsui, Benjamin; Abraham, M. Roselle

    2013-01-01

    Rationale Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known which factors, other than cell number can influence the molecular imaging signal obtained from reporter genes. Objective The effects of cell dissociation/suspension on cellular bioenergetics and the signal obtained by firefly luciferase(fluc) and human Na-I symporter(hNIS) labeling of cardiosphere-derived cells (CDCs) was investigated. Methods and Results 18FDG uptake, ATP levels, 99mTc-pertechnetate uptake and bioluminescence were measured in vitro, in adherent and suspended CDCs. In vivo dual isotope SPECT-CT imaging or bioluminescence imaging (BLI) were performed 1hr and 24hrs following CDC transplantation. SPECT quantification was performed using a phantom for signal calibration. Cell loss between 1hr & 24hrs post-transplantation was quantified by qPCR and ex vivo luciferase assay. Cell dissociation followed by suspension for 1hr resulted in decreased glucose uptake, cellular ATP, 99mTc uptake and BLI signal by 82%, 43%, 42%, and 44% respectively, when compared to adherent cells, in vitro. In vivo 99mTc uptake was significantly lower at 1hr, when compared to 24hrs following cell transplantation in the non-infarct (p<0.001, n=3) and infarct (p<0.001, n =4) model, despite significant cell loss during this period. The in vivo BLI signal was significantly higher at 1hr than at 24hrs (p<0.01), with the BLI signal being higher when CDCs were suspended in glucose-containing medium compared to saline(PBS). Conclusion Adhesion is an important determinant of cellular bioenergetics, 99mTc-pertechnetate uptake and BLI signal. BLI and NIS imaging may be useful for in vivo optimization of bioenergetics in transplanted cells. PMID:23255420

  17. Searching for Extant Life on Mars - The ATP-Firefly LuciferinLuciferase Technique

    NASA Astrophysics Data System (ADS)

    Obousy, R. K.; Tziolas, A. C.; Kaltsas, K.; Sims, M. R.; Grant, W. D.

    We have investigated the use of the ATP-Firefly Luciferin/Luciferase (FFL) enzymic photoluminescent reaction as a possible means of detecting extant life in the Martian environment. Experiments carried out by the authors illustrate the capacity of the method to successfully detect extant forms of life on Mars assuming ATP is an intrinsic part of the biochemistry of such life-forms. A photodiode based apparatus, built to test the assumptions and applicability of the ATP-Firefly Luciferase/Luciferin technique to an exobiologically inclined mission to Mars, revealed the adequate resolution and reproducibility of the methodology plus areas of improvement. Also detailed are extraction, delivery and analysis system concepts, proposed for future Mars missions.

  18. Near infrared bioluminescence resonance energy transfer from firefly luciferase--quantum dot bionanoconjugates.

    PubMed

    Alam, Rabeka; Karam, Liliana M; Doane, Tennyson L; Zylstra, Joshua; Fontaine, Danielle M; Branchini, Bruce R; Maye, Mathew M

    2014-12-12

    The bioluminescence resonance energy transfer (BRET) between firefly luciferase enzymes and semiconductive quantum dots (QDs) with near infrared emission is described. The QD were phase transferred to aqueous buffers using a histidine mediated phase transfer route, and incubated with a hexahistidine tagged, green emitting variant of firefly luciferase from Photinus pyralis (PPyGRTS). The PPyGRTS were bound to the QD interface via the hexahistidine tag, which effectively displaces the histidine layer and binds directly to the QD interfaces, allowing for short donor-acceptor distances (∼5.5 nm). Due to this, high BRET efficiency ratios of ∼5 were obtained. These PPyGRTS-QD bio-nano conjugates were characterized by transmission electron microscopy, thermal gravimetric analysis, Fourier transform infrared spectroscopy and BRET emission studies. The final optimized conjugate was easily observable by night vision imaging, demonstrating the potential of these materials in imaging and signaling/sensing applications. PMID:25414169

  19. Enhancer/Promoter Activities of the Long/Middle Wavelength-Sensitive Opsins of Vertebrates Mediated by Thyroid Hormone Receptor β2 and COUP-TFII

    PubMed Central

    Iida, Atsumi; Itoh, Toshio; Watanabe, Sumiko

    2013-01-01

    Cone photopigments (opsins) are crucial elements of, and the first detection module in, color vision. Individual opsins have different wavelength sensitivity patterns, and the temporal and spatial expression patterns of opsins are unique and stringently regulated. Long and middle wavelength-sensitive (L/M) opsins are of the same phylogenetic type. Although the roles of thyroid hormone/TRß2 and COUP-TFs in the transcriptional regulation of L/M opsins have been explored, the detailed mechanisms, including the target sequence in the enhancer of L/M opsins, have not been revealed. We aimed to reveal molecular mechanisms of L/M opsins in vertebrates. Using several human red opsin enhancer/promoter-luciferase reporter constructs, we found that TRß2 increased luciferase activities through the 5′-UTR and intron 3–4 region, whereas the presence of T3 affected only the intron 3–4 region-dependent luciferase activity. Furthermore, COUP-TFII suppressed intron 3–4 region-dependent luciferase activities. However, luciferase expression driven by the mouse M opsin intron 3–4 region was only slightly increased by TRß2, and rather enhanced by COUP-TFII. To determine whether these differential responses reflect differences between primates and rodents, we examined the enhancer/promoter region of the red opsin of the common marmoset. Interestingly, while TRß2 increased 5′-UTR- or intron 3–4 region-driven luciferase expression, as observed for the human red opsin, expression of the latter luciferase was not suppressed by COUP-TFII. In fact, immunostaining of common marmoset retinal sections revealed expression of COUP-TFII and red opsin in the cone cells. PMID:24058409

  20. Imaging Tumor Vascularity and Response to Anti-Angiogenic Therapy Using Gaussia Luciferase.

    PubMed

    Kantar, Rami S; Lashgari, Ghazal; Tabet, Elie I; Lewandrowski, Grant K; Carvalho, Litia A; Tannous, Bakhos A

    2016-01-01

    We developed a novel approach to assess tumor vascularity using recombinant Gaussia luciferase (rGluc) protein and bioluminescence imaging. Upon intravenous injection of rGluc followed by its substrate coelenterazine, non-invasive visualization of tumor vascularity by bioluminescence imaging was possible. We applied this method for longitudinal monitoring of tumor vascularity in response to the anti-angiogenic drug tivozanib. This simple and sensitive method could be extended to image blood vessels/vasculature in many different fields. PMID:27198044

  1. Bioluminescent microassay of various metabolites using bacterial luciferase co-immobilized with multienzyme systems.

    PubMed

    Ugarova, N N; Lebedeva, O V; Frumkina, I G

    1988-09-01

    Co-immobilization methods have been developed for a bienzymatic system of luminescent Beneckea harveyi bacteria with formate dehydrogenase, glucose-6-phosphate dehydrogenase, and phosphoglucomutase. Bioluminescent assays have been devised for NADH, NAD, FMN, glucose 6-phosphate, and glucose 1-phosphate using the co-immobilized enzyme preparation. The lowest detection limits were in the picomole range with the bacterial extract and in the femtomole range with the partially purified enzymes, bacterial luciferase, and NADH:FMN oxidoreductase. PMID:3263818

  2. Imaging Tumor Vascularity and Response to Anti-Angiogenic Therapy Using Gaussia Luciferase

    PubMed Central

    Kantar, Rami S.; Lashgari, Ghazal; Tabet, Elie I.; Lewandrowski, Grant K.; Carvalho, Litia A.; Tannous, Bakhos A.

    2016-01-01

    We developed a novel approach to assess tumor vascularity using recombinant Gaussia luciferase (rGluc) protein and bioluminescence imaging. Upon intravenous injection of rGluc followed by its substrate coelenterazine, non-invasive visualization of tumor vascularity by bioluminescence imaging was possible. We applied this method for longitudinal monitoring of tumor vascularity in response to the anti-angiogenic drug tivozanib. This simple and sensitive method could be extended to image blood vessels/vasculature in many different fields. PMID:27198044

  3. Detection of luciferase gene sequence in nonluminescent Vibrio cholerae by colony hygridization and polymerase chain reaction

    SciTech Connect

    Palmer, L.M.; Colwell, R.R. )

    1991-05-01

    Bioluminescence is a trait observed among approximately 10% of Vibrio cholerae isolates. We have demonstrated that not only do some strains of V. cholerae produce low levels of light, undetectable by the human eye, but the luciferase gene sequence is present in strains of V. cholerae which emit no detectable light, evidenced by hybridization with a luciferase DNA probe. Comparisons of the amino acid sequences of luciferase regions of amino acid identity. The polymerase chain reaction method of DNA amplification with oligonucleotide primers based on these regions was used to isolate a region of the luxA gene from both luminescent and nonluminescent V. cholerae strains. The nucleotide sequence of this region was determined and reveals that nonluminescent V. cholerae have 99.7% nucleotide sequence similarity in this region with the luminescent biovar V. cholerae by albensis as well as significant similarity to other species of bioluminescent bacteria, a finding that is in accord with the hypothesis that these species have a common luminescent ancestor, most probably from the marine environment.

  4. Novel mammalian cell lines expressing reporter genes for the detection of environmental chemicals activating endogenous aryl hydrocarbon receptors (ArhR) or estrogen receptors (ER).

    PubMed

    Minh, Si Do; Below, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2008-12-01

    We have constructed two vector systems (pDMS5, pSAB2) containing the promoter regions of the human CYP1A1 gene including xenobiotic response elements or the promoter region of the Xenopus laevis vitellogenin A2 gene including estrogen response elements, respectively, and the genes for green fluorescent protein and firefly luciferase. These vectors were transfected into CHO-K1 cells. Transiently transfected cells consistently responded to 1 nmol/l TCDD (dioxin) or 10 nmol/l 17ss-estradiol, respectively, with a 3-5 fold increase in luciferase activity. Permanent cell lines were selected by culturing transiently transfected cells under continued presence of antibiotics and dilution cloning. Cells which had stably integrated the vector-DNA into the genomic DNA were selected. SiF6 cells responded to treatment with TCDD, PCB126, benzo(a)pyrene or indirubin-3'-monoxime in the concentration range between 0 and 1 micromol/l. SiG12 cells responded to treatment with bisphenol A, 4-MBC and DDT in the concentration range between 0 and 10 micromol/l. Compared with the controls, luciferase mRNA-abundance (semi-quantitative RT-PCR) and luciferase activity (luminescence assay) were elevated up to 3-fold. Resveratrol or tamoxifen, respectively, worked as full antagonists. Luciferase expression was increased upon treatment of cells with extracts of spiked soil samples indicating that our systems are suited for screening of environmental samples. PMID:18835349

  5. Needs assessment activity report: Fiscal year 1995

    SciTech Connect

    1995-09-01

    The Needs Assessment program has assessed the packaging requirements of many U.S. Department of Energy (DOE) sites. These assessments have involved site visits and meetings with personnel involved with transportation and packaging of hazardous materials. By September 1995, 24 DOE facilities had been visited, with 14 site visits occurring in fiscal year 1995. As a result, these sites have been informed of some of the packaging activities that DOE has sponsored and is sponsoring, have been apprised of the affects of upcoming changes to transportation regulations, have discussed their near-term packaging needs, and have shared unique packaging they have developed, which may be of use to other DOE facilities. Program successes include discovery of a need for a reusable Type A liquid sample packaging and its development within another DOE task and establishing communications pathways between DOE sites that have similar transportation and packaging needs. This report recommends that the Needs Assessment activity continue to pursue the strategy of visiting DOE sites to meet with their transportation and packaging personnel. These visits will ensure that DOE needs are met, communications pathways between DOE sites are established and cultivated, and redundant packaging development is identified. The site visits should be expanded to include meetings with the long-range and strategic planners at each site, and at the DOE-Headquarters level, to ensure that all future transportation and packaging needs are identified early enough to allow adequate transportation assessment and packaging development. This activity could become a permanent conduit for information and will ensure that all future DOE transportation and packaging needs are satisfied in a cost-effective, timely, and efficient manner.

  6. 24 CFR 1710.310 - Annual report of activity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 5 2011-04-01 2011-04-01 false Annual report of activity. 1710.310... § 1710.310 Annual report of activity. (a) As an integral part of the Statement of Record, the developer shall file with the Secretary an Annual Report of Activity on any initial or consolidated...

  7. 24 CFR 1710.310 - Annual report of activity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 5 2013-04-01 2013-04-01 false Annual report of activity. 1710.310... § 1710.310 Annual report of activity. (a) As an integral part of the Statement of Record, the developer shall file with the Secretary an Annual Report of Activity on any initial or consolidated...

  8. 24 CFR 1710.310 - Annual report of activity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 5 2014-04-01 2014-04-01 false Annual report of activity. 1710.310... § 1710.310 Annual report of activity. (a) As an integral part of the Statement of Record, the developer shall file with the Secretary an Annual Report of Activity on any initial or consolidated...

  9. Promoter-directed expression of recombinant fire-fly luciferase in the salivary glands of Hermes-transformed Aedes aegypti.

    PubMed

    Coates, C J; Jasinskiene, N; Pott, G B; James, A A

    1999-01-21

    Molecular genetic analyses of biological properties characteristic of insect vectors of disease, such as hematophagy and competence for pathogens, require the ability to isolate and characterize genes involved in these processes. We have been working to develop molecular approaches for studying the promoter function of genes that are expressed specifically in the adult salivary glands of the yellow fever mosquito, Aedes aegypti. Genomic DNA fragments containing cis-acting promoter elements from the Maltase-like I (MalI) and Apyrase (Apy) genes were cloned so as to direct the expression of the reporter gene, luciferase (luc). The function of the promoters was assayed transiently in cultured insect cells and by germ-line transformation of Ae. aegypti. MalI and Apy DNA fragments consisting of at least 650 nucleotides (nt) of DNA immediately adjacent to the 5'-end of the initiation codon of the mosquito genes directed constitutive expression of the luc reporter gene in cultured cells. When introduced into Ae. aegypti chromosomes, approximately 1.5 kilobases (kb) of each promoter were able to direct the predicted developmental-, sex- and tissue-specific expression of the reporter gene in patterns identical to those determined for the respective endogenous genes. PMID:9931506

  10. Thermostable luciferase from Luciola cruciate for imaging of carbon nanotubes and carbon nanotubes carrying doxorubicin using in vivo imaging system.

    PubMed

    El-Sayed, Ramy; Eita, Mohamed; Barrefelt, Asa; Ye, Fei; Jain, Himanshu; Fares, Mona; Lundin, Arne; Crona, Mikael; Abu-Salah, Khalid; Muhammed, Mamoun; Hassan, Moustapha

    2013-04-10

    In the present study, we introduce a novel method for in vivo imaging of the biodistribution of single wall carbon nanotubes (SWNTs) labeled with recombinant thermo-stable Luciola cruciata luciferase (LcL). In addition, we highlight a new application for green fluorescent proteins in which they are utilized as imaging moieties for SWNTs. Carbon nanotubes show great positive potential compared to other drug nanocarriers with respect to loading capacity, cell internalization, and biodegradability. We have also studied the effect of binding mode (chemical conjugation and physical adsorption) on the chemiluminescence activity, decay rate, and half-life. We have shown that through proper chemical conjugation of LcL to CNTs, LcL remained biologically active for the catalysis of d-luciferin in the presence of ATP to release detectable amounts of photons for in vivo imaging. Chemiluminescence of LcL allows imaging of CNTs and their cargo in nonsuperficial locations at an organ resolution with no need of an excitation source. Loading LcL-CNTs with the antitumor antibiotic doxorubicin did not alter their biological activity for imaging. In vivo imaging of LcL-CNTs has been carried out using "IVIS spectrum" showing the uptake of LcL-CNTs by different organs in mice. We believe that the LcL-CNT system is an advanced powerful tool for in vivo imaging and therefore a step toward the advancement of the nanomedicine field. PMID:23520995

  11. Field Operations Program Activities Status Report

    SciTech Connect

    J. E. Francfort; D. V. O'Hara; L. A. Slezak

    1999-05-01

    The Field Operations Program is an electric vehicle testing and evaluation program sponsored by US Department of Energy and managed by the Idaho National Engineering and Environmental Laboratory. The Program's goals are to evaluate electric vehicles in real-world applications and environments, support electric vehicle technology advancement, develop infrastructure elements necessary to support significant electric vehicle use, support increased use of electric vehicles in federal fleets, and increase overall awareness and acceptance of electric vehicles. This report covers Program activities from fiscal year 1997 through mid-fiscal year 1999. The Field Operations Program succeeded the Site Operator Program, which ended in September 1996. Electric vehicle testing conducted by the Program includes baseline performance testing (EV America testing), accelerated reliability (life-cycle) testing, and fleet testing. The baseline performance parameters include accelerations, braking, range, energy efficiency, and charging time. The Program collects accelerated reliability and fleet operations data on electric vehicles operated by the Program's Qualified Vehicle Testing (QVT) partners. The Program's QVT partners have over 3 million miles of electric vehicle operating experience.

  12. Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells.

    PubMed

    Heredia-Rojas, J Antonio; Rodríguez de la Fuente, Abraham Octavio; Alcocer González, Juan Manuel; Rodríguez-Flores, Laura E; Rodríguez-Padilla, Cristina; Santoyo-Stephano, Martha A; Castañeda-Garza, Esperanza; Taméz-Guerra, Reyes S

    2010-10-01

    It has been reported that 50-60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 μT on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line. PMID:20835776

  13. Measurements of serum glucose using the luciferin/Luciferase system and a liquid scintillation spectrometer

    SciTech Connect

    Idahl, L.A.; Sandstroem, P.E.; Sehlin, J.

    1986-05-15

    A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.

  14. NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

    PubMed Central

    Boute, Nicolas; Lowe, Peter; Berger, Sven; Malissard, Martine; Robert, Alain; Tesar, Michael

    2016-01-01

    Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings. PMID:26924984

  15. NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening.

    PubMed

    Boute, Nicolas; Lowe, Peter; Berger, Sven; Malissard, Martine; Robert, Alain; Tesar, Michael

    2016-01-01

    Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a "swiss army knife solution" for today and future high throughput antibody drug screenings. PMID:26924984

  16. 1990 Activity report for 1986-1992

    SciTech Connect

    Cantwell, K.

    1996-01-01

    As discussed in last year`s Activity Report, a fairly complete analysis of SPEAR problems was performed in concert with SLAC, and a major maintenance/improvement process was initiated in the spring of 1989. This run made it apparent that SPEAR will remain a very useful and impressive synchrotron radiation storage ring for at least a decade, given a steady maintenance and improvement program. More details about SPEAR functioning during the run, as well as SPEAR improvements, are contained in Chapter I. The formal SPEAR injector construction project was completed in November, 1990, on-time and on-budget. Although DOE was not able to provide anticipated FY90 commissioning funds, preliminary commissioning was performed and 2.3 GeV injection to SPEAR was demonstrated. A discussion of the Injector project is contained in Chapter II. Commissioning of the injector and the injector/SPEAR complex is continuing in 1991 with Users participating during the May-September period. This user participation allowed normal experimentation, so that systems could be tested critically, but with the commissioning process having higher priority than data acquisition. Another major event in 1990 was the full dedication of SPEAR to the synchrotron radiation program. Previously SPEAR was considered a high energy physics machine that was partially dedicated to synchrotron radiation. The full dedication means that the accelerator can be modified and improved for synchrotron radiation research. Despite the heavy emphasis on completing the Injector, many beam line improvements were achieved, as described in Chapter IV. Among these was the optimization of stations 6-2 and 10-2, the provision of a considerably larger hutch for Station 1-5, which contains the area detector diffractometer, and the introduction of white light capability on 10-2. The provision of good beam during the month of April made an appreciable amount of experimentation possible. These and other runs are described in Chapter VI.

  17. Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System▿

    PubMed Central

    Zhang, Zhen; Rowe, Jenny; Wang, Weijia; Sommer, Marvin; Arvin, Ann; Moffat, Jennifer; Zhu, Hua

    2007-01-01

    To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo. PMID:17581997

  18. National Synchrotron Light Source 2010 Activity Report

    SciTech Connect

    Rowe, M.; Snyder, K. J.

    2010-12-29

    This is a very exciting period for photon sciences at Brookhaven National Laboratory. It is also a time of unprecedented growth for the Photon Sciences Directorate, which operates the National Synchrotron Light Source (NSLS) and is constructing NSLS-II, both funded by the Department of Energy's Office of Science. Reflecting the quick pace of our activities, we chose the theme 'Discovery at Light Speed' for the directorate's 2010 annual report, a fiscal year bookended by October 2009 and September 2010. The year began with the news that NSLS users Venki Ramakrishnan of Cambridge University (also a former employee in Brookhaven's biology department) and Thomas A. Steitz of Yale University were sharing the 2009 Nobel Prize in Chemistry with Ada E. Yonath of the Weizmann Institute of Science. Every research project has the potential for accolades. In 2010, NSLS users and staff published close to 900 papers, with about 170 appearing in premiere journals. Those are impressive stats for a facility nearly three decades old, testament to the highly dedicated team keeping NSLS at peak performance and the high quality of its user community. Our NSLS users come from a worldwide community of scientists using photons, or light, to carry out research in energy and environmental sciences, physics, materials science, chemistry, biology and medicine. All are looking forward to the new capabilities enabled by NSLS-II, which will offer unprecedented resolution at the nanoscale. The new facility will produce x-rays more than 10,000 times brighter than the current NSLS and host a suite of sophisticated instruments for cutting-edge science. Some of the scientific discoveries we anticipate at NSLS-II will lead to major advances in alternative energy technologies, such as hydrogen and solar. These discoveries could pave the way to: (1) catalysts that split water with sunlight for hydrogen production; (2) materials that can reversibly store large quantities of electricity or hydrogen; (3

  19. Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

    PubMed

    Decock, Marie; El Haylani, Laetitia; Stanga, Serena; Dewachter, Ilse; Octave, Jean-Noël; Smith, Steven O; Constantinescu, Stefan N; Kienlen-Campard, Pascal

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the β-amyloid peptide (Aβ) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aβ production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and β C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aβ production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing. PMID:26500837

  20. Recovery Efficiency Test Project: Phase 1, Activity report

    SciTech Connect

    Overbey, W.K. Jr.; Wilkins, D.W.; Keltch, B.; Saradji, B.; Salamy, S.P.

    1988-04-01

    This report is the second volume of the Recovery Efficiency Test Phase I Report of Activities. Volume 1 covered selection, well planning, drilling, coring, logging and completion operations. This volume reports on well testing activities, reclamation activities on the drilling site and access roads, and the results of physical and mechanical properties tests on the oriented core material obtained from a horizontal section of the well. 3 refs., 21 figs., 10 tabs.

  1. Advanced Light Source Activity Report 2002

    SciTech Connect

    Duque, Theresa; Greiner, Annette; Moxon, Elizabeth; Robinson, Arthur; Tamura, Lori

    2003-06-12

    This annual report of the Advanced Light Source details science highlights and facility improvements during the year. It also offers information on events sponsored by the facility, technical specifications, and staff and publication information.

  2. NATIONAL SYNCHROTRON LIGHT SOURCE ACTIVITY REPORT 1998.

    SciTech Connect

    ROTHMAN,E.

    1999-05-01

    In FY 1998, following the 50th Anniversary Year of Brookhaven National Laboratory, Brookhaven Science Associates became the new Managers of BNL. The new start is an appropriate time to take stock of past achievements and to renew or confirm future goals. During the 1998 NSLS Annual Users Meeting (described in Part 3 of this Activity Report), the DOE Laboratory Operations Board, Chaired by the Under Secretary for Energy, Ernest Moniz met at BNL. By chance all the NSLS Chairmen except Martin Blume (acting NSLS Chair 84-85) were present as recorded in the picture. Under their leadership the NSLS has improved dramatically: (1) The VUV Ring current has increased from 100 mA in October 1982 to nearly 1 A today. For the following few years 10 Ahrs of current were delivered most weeks - NSLS now exceeds that every day. (2) When the first experiments were performed on the X-ray ring during FY1985 the electron energy was 2 GeV and the current up to 100 mA - the X-Ray Ring now runs routinely at 2.5 GeV and at 2.8 GeV with up to 350 mA of current, with a very much longer beam half-life and improved reliability. (3) Starting in FY 1984 the proposal for the Phase II upgrade, mainly for a building extension and a suite of insertion devices and their associated beamlines, was pursued - the promises were delivered in full so that for some years now the NSLS has been running with two undulators in the VUV Ring and three wigglers and an undulator in the X-Ray Ring. In addition two novel insertion devices have been commissioned in the X13 straight. (4) At the start of FY 1998 the NSLS welcomed its 7000th user - attracted by the opportunity for pursuing research with high quality beams, guaranteed not to be interrupted by 'delivery failures', and welcomed by an efficient and caring user office and first class teams of PRT and NSLS staff. R & D have lead to the possibility of running the X-Ray Ring at the higher energy of 2.8 GeV. Figure 1 shows the first user beam, which was provided

  3. NATIONAL SYNCHROTRON LIGHT SOURCE ACTIVITY REPORT 2004

    SciTech Connect

    MILLER,L.

    2005-05-01

    The NSLS remains a viable and productive facility, as can be seen by the rich and diverse science produced in 2004. In one of these exciting research projects published in Nature, researchers detected a rare 'hole crystal' in a cuprate superconductor, which may provide insight into high-temperature superconductivity. In another Nature publication, the crystal structure of a segment of RNA was determined, opening a new window of knowledge into that crucial molecule. These are just a couple of the science highlights of 2004, and many others are displayed in the pages of this report. All told, more than 700 publications resulted from NSLS research this year, the facility hosted 2,299 users, and the number of experiments performed rose from 1,145 in 2003 to 1,374 nuclear indications that the NSLS continues to thrive. As the NSLS accelerator complex enters its third decade of operations, it continues to perform very well. For 2004, the overall reliability of the VUV-IR ring was excellent at 99 percent. The reliability of the x-ray ring was just shy of 92 percent, primarily due to the need to replace the injection septum vacuum chamber, which developed a leak during the middle of the year. The Operations Division did a tremendous job of installing our spare chamber in minimal time, despite the complexity of the job and the inaccessibility of its location in the ring, as well as keeping downtime to a minimum throughout the rest of the year. In order to continue to meet the needs of users, several key beamline upgrades took place this year that will enrich our scientific programs, including upgrades to beamlines U12IR, X1A, X13A, and X21. We are very excited about two brand-new beamlines that were commissioned in 2004: X29 and X27A. X29 is the new mini-gap undulator beamline designed for macromolecular crystallography, and it will meet the growing demand of NSLS users who perform research in that area. The establishment of an x-ray microprobe at beamline X27A, optimized

  4. Phytochemicals Mediate the Expression and Activity of OCTN2 as Activators of the PPARγ/RXRα Pathway

    PubMed Central

    Luo, Jian; Qu, Jian; Yang, Rui; Ge, Meng-Xue; Mei, Yin; Zhou, Bo-Ting; Qu, Qiang

    2016-01-01

    Many phytochemicals exert activities as agonists of peroxisome proliferator-activated receptor gamma (PPARγ). This study aims to investigate whether phytochemicals are agonists of the PPARγ/RXRα pathway and modulate the target gene OCTN2. In this study, a luciferase reporter gene system was used to screen novel OCTN2 activators from 39 phytochemicals. Kaempferol, curcumin, and puerarin were found to show the significant PPRE-mediated luciferase activities (>150%) at 20 μM and showed a dose-dependent manner. Phytochemicals also elevated the mRNA and protein expression of OCTN2 in a dose-dependent fashion in colorectal cancer SW480 cells. These induction effects were gradually inhibited by PPARγ antagonist GW9662 in the luciferase reporter gene system and in SW480 cells. Moreover, the results of cell viability assay imply that three phytochemicals probably induce OCTN2 expression leading to the enhanced uptake of its substrate, oxaliplatin, thereby making cells more sensitive to oxaliplatin. The molecular docking study showed the possible binding sites of phytochemicals in PPARγ protein, and all of the docked phytochemicals fitted the same active pocket in PPARγ as troglitazone. All three phytochemicals exhibited hydrogen bonds between their polar moieties and the amino acid residues. Thus, we identified three phytochemicals as PPARγ ligands, which potentiated the expression and activity of OCTN2. PMID:27445823

  5. REPORT ON ACTIVITIES, 1964-1966.

    ERIC Educational Resources Information Center

    FROIMSON, MARCIA; MAX, DAVID

    A REPORT ISSUED BY THE ISRAELI NATIONAL INSTITUTE FOR RESEARCH IN THE BEHAVIORAL SCIENCES INDICATES THAT MANY OF THEIR CURRENT PROJECTS ARE INVESTIGATING CULTURALLY DEPRIVED ISRAELI CHILDREN. TOPICS INCLUDE THE GROWTH AND DEVELOPMENT, LEARNING PSYCHOLOGY, AND FAMILY AND KINDERGARTEN BACKGROUNDS OF THESE CHILDREN AS WELL AS THE PREPARATION OF…

  6. [Electron transfer activation of thiophene]. Progress report

    SciTech Connect

    Rauchfuss, T.B.

    1993-09-01

    This report is divided into: reduction of coordinated thiophenes,protonation of reduced thiophene complexes, bimetallic thiophene desulfurization, thermal decomposition of {eta}{sup 4}-thiophene complexes, metal ion-promoted hydrolysis of thiophenes, thermal fragmentation of hydrolyzed thiophene ligands, and organic chemistry of hydrolyzed thiophene ligands.

  7. College-School Collaborative Activities Report.

    ERIC Educational Resources Information Center

    Maryland State Higher Education Commission, Annapolis.

    This report inventories academic and administrative projects which are collaborative ventures between higher education institutions and elementary and secondary schools in Maryland. Fourteen community colleges, 9 public four-year campuses, 2 of the University System of Maryland extension services, and 16 independent institutions (including all of…

  8. 12 CFR 1806.201 - Measuring and reporting Qualified Activities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 7 2010-01-01 2010-01-01 false Measuring and reporting Qualified Activities. 1806.201 Section 1806.201 Banks and Banking COMMUNITY DEVELOPMENT FINANCIAL INSTITUTIONS FUND, DEPARTMENT OF THE TREASURY BANK ENTERPRISE AWARD PROGRAM Awards § 1806.201 Measuring and reporting Qualified Activities. (a) General. An Applicant...

  9. 10 CFR 490.507 - Credit activity reporting requirements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 false Credit activity reporting requirements. 490.507 Section 490.507 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ALTERNATIVE FUEL TRANSPORTATION PROGRAM Alternative Fueled Vehicle Credit Program § 490.507 Credit activity reporting requirements. (a) A...

  10. 10 CFR 490.507 - Credit activity reporting requirements.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 3 2011-01-01 2011-01-01 false Credit activity reporting requirements. 490.507 Section 490.507 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ALTERNATIVE FUEL TRANSPORTATION PROGRAM Alternative Fueled Vehicle Credit Program § 490.507 Credit activity reporting requirements. (a) A...

  11. 10 CFR 490.507 - Credit activity reporting requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Credit activity reporting requirements. 490.507 Section 490.507 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ALTERNATIVE FUEL TRANSPORTATION PROGRAM Alternative Fueled Vehicle Credit Program § 490.507 Credit activity reporting requirements. (a) A...

  12. 24 CFR 81.63 - Annual Housing Activities Report.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Annual Housing Activities Report. 81.63 Section 81.63 Housing and Urban Development Office of the Secretary, Department of Housing and... Annual Housing Activities Report. To comply with the requirements in sections 309(n) of the Fannie...

  13. 33 CFR 150.835 - Reporting sabotage or subversive activity.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 2 2014-07-01 2014-07-01 false Reporting sabotage or subversive... Reporting sabotage or subversive activity. The owner, operator, or person in charge of a deepwater port must... possible means, any evidence of sabotage or subversive activity against any vessel at the deepwater port...

  14. 33 CFR 150.835 - Reporting sabotage or subversive activity.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 2 2013-07-01 2013-07-01 false Reporting sabotage or subversive... Reporting sabotage or subversive activity. The owner, operator, or person in charge of a deepwater port must... possible means, any evidence of sabotage or subversive activity against any vessel at the deepwater port...

  15. Ab initio quantum-chemical study on emission spectra of bioluminescent luciferases by fragment molecular orbital method

    NASA Astrophysics Data System (ADS)

    Tagami, Ayumu; Ishibashi, Nobuhiro; Kato, Dai-ichiro; Taguchi, Naoki; Mochizuki, Yuji; Watanabe, Hirofumi; Ito, Mika; Tanaka, Shigenori

    2009-04-01

    Bioluminescence spectra of firefly Luciola cruciata were theoretically analyzed on the basis of the fragment molecular orbital (FMO) method. The CIS(D) and PR-CIS(Ds) methods were employed for the calculations of emission energies of wild-type and mutant luciferase-oxyluciferin systems, and various multi-layer FMO calculations were performed changing the sizes of the luciferase protein and of the chromophore to which the excited-state calculations were applied. We have thus reproduced the experimental emission energies of wild-type and mutant luciferase systems with good accuracy, which provides useful information concerning the roles of protein environment for the color tuning of the bioluminescence spectra of firefly.

  16. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10{sup 9} PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

  17. Mathemagenic Activities Program: [Reports on Constructivism].

    ERIC Educational Resources Information Center

    Smock, Charles D., Ed.

    This set of five papers is related to the Mathemagenic Activities Program (MAP) for early childhood education of the University of Georgia's Follow Through Program. The MAP is based on Piagetian theory and provides sequentially structured sets of curriculum materials and processes that are designed to continually challenge children to learn about…

  18. Effect of cadmium on lake water bacteria as determined by the luciferase assay of adenosine triphosphate

    SciTech Connect

    Seyfried, P.L.; Horgan, C.B.L.

    1981-10-01

    A firefly luciferase assay of bacterial adenosine triphosphate (ATP) was developed to measure the toxic effects of cadmium ions on aquatic organisms. Toxicity was monitored using intracellular (I/C) ATP (in micrograms per litre) as well as plate counts (colony-forming units per millilitre). The bacteria, which belonged mainly to the families Enterobacteriaceae and Pseudomonadaceae, exhibited varying degrees of resistance to up to 100 ppm cadmium when grown in a glucose-salts medium at pH 6.8. Among the organisms tested, cadmium resistance decreased in the following order: Pseudomonas vesicularis > P. aeruginosa > Enterobacter sp. > P. fluorescens > Chromobacter sp. > Serratia sp. A rise in the pH of the growth medium from 5 to 7 resulted in increased toxicity of cadmium.

  19. A real-time kinetic study of luciferase inactivation by pulsed ionizing radiation

    SciTech Connect

    Bell, D.H.; Gould, J.M.; Patterson, L.K.

    1982-06-01

    The real-time kinetics of radiation-induced inactivation of the luminescent firefly luciferase-luciferin system were investigated. A single, microsecond pulse from a Van de Graaff accelerator delivered to the system is sufficient to decrease the luminescence by over 60%. This decrease exhibits exponential behavior and has a half-time of 46 +/- 6 msec. In both steady-state and pulsed studies, the dose dependence of the inactivation is independent of the dose rate. Likewise, the decay kinetics are independent of the dose per pulse. These studies suggest that the enzyme is altered in a way that inteferes with the initial steps of catalysis without affecting the subsequent steps which lead to light emission.

  20. Individual subunits of bacterial luciferase are molten globules and interact with molecular chaperones.

    PubMed Central

    Flynn, G C; Beckers, C J; Baase, W A; Dahlquist, F W

    1993-01-01

    We have studied the assembly of a large heterodimeric protein, bacterial luciferase, by mixing purified subunits expressed separately in bacteria. The individual subunits alpha and beta contain much (66% and 50%, respectively) of the alpha-helix content of the native heterodimer as measured by circular dichroism, yet the alpha subunit lacks observable tertiary structure as measured by NMR. These results are consistent with the alpha subunit existing in a molten globule or collapsed form prior to assembly. The molecular chaperone GroEL binds reversibly to both subunits prior to assembly. Since these observations were obtained under physiological conditions, we propose that the molten globule exists as a stable form during folding or assembly in the cell. Either the molten globule form of the subunits is an authentic folding intermediate or it is in rapid equilibrium with one. GroEL may function by facilitating assembly through stabilization of these incompletely folded subunits. Images Fig. 4 PMID:7902573

  1. 33 CFR 150.835 - Reporting sabotage or subversive activity.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Reporting sabotage or subversive... Reporting sabotage or subversive activity. The owner, operator, or person in charge of a deepwater port must immediately report to the Captain of the Port, by the fastest possible means, any evidence of sabotage...

  2. 33 CFR 150.835 - Reporting sabotage or subversive activity.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 2 2012-07-01 2012-07-01 false Reporting sabotage or subversive... Reporting sabotage or subversive activity. The owner, operator, or person in charge of a deepwater port must immediately report to the Captain of the Port, by the fastest possible means, any evidence of sabotage...

  3. Design for manufacturability production management activity report

    NASA Astrophysics Data System (ADS)

    Miyazaki, Norihiko; Sato, T.; Honma, M.; Yoshioka, N.; Hosono, K.; Onodera, T.; Itoh, H.; Suzuki, H.; Uga, T.; Kadota, K.; Iriki, N.

    2006-05-01

    Design For Manufacturability Production Management (DFM-PM) Subcommittee has been started in succession to Reticle Management Subcommittee (RMS) in Semiconductor Manufacturing Technology Committee for Japan (SMTCJ) from 2005. Our activity focuses on the SoC (System On Chip) Business, and it pursues the improvement of communication in manufacturing technique. The first theme of activity is the investigation and examination of the new trends about production (manufacturer) technology and related information, and proposals of business solution. The second theme is the standardization activity about manufacture technology and the cooperation with related semiconductors' organizations. And the third theme is holding workshop and support for promotion and spread of the standardization technology throughout semiconductor companies. We expand a range of scope from design technology to wafer pattern reliability and we will propose the competition domain, the collaboration area and the standardization technology on DFM. Furthermore, we will be able to make up a SoC business model as the 45nm node technology beyond manufacturing platform in cooperating with the design information and the production information by utilizing EDA technology.

  4. Hypoxia-sensitive reporter system for high-throughput screening.

    PubMed

    Tsujita, Tadayuki; Kawaguchi, Shin-ichi; Dan, Takashi; Baird, Liam; Miyata, Toshio; Yamamoto, Masayuki

    2015-01-01

    The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS. PMID:25746387

  5. Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: comparison of substrate specificity for C2-modified coelenterazines.

    PubMed

    Inouye, Satoshi; Sahara-Miura, Yuiko; Sato, Jun-ichi; Iimori, Rie; Yoshida, Suguru; Hosoya, Takamitsu

    2013-03-01

    The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%)>RLase (8.0%)>RLase-547 (0.73%)>19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases. PMID:23274053

  6. Needs assessment activity report: April 1995

    SciTech Connect

    1995-04-01

    As part of a US Department of Energy Headquarters task (DOE-HQ), the Packaging Operations and Development Group within Westinghouse Hanford Company (WHC) has assessed the packaging needs of many DOE sites. These assessments have involved site visits and meetings with personnel involved with transportation and packaging of hazardous materials. By March 1995, 20 DOE facilities had been visited. As a result, these sites been informed of some of the packaging activities that DOE has sponsored and is sponsoring, have been apprised of the affects of upcoming changes to transportation regulations, have discussed their short-term packaging needs, and have shared unique packaging they have developed which may be of use to other DOE facilities. Program successes include discovery of a need for a reusable Type A liquid sample packaging and its development within another DOE task, establishing communications pathways between DOE sites that have similar transportation and packaging needs, and starting to establish a centralized packaging clearinghouse that will coordinate DOE Complex needs and improve the cost-effectiveness of transportation and packaging activities.

  7. Evaluation of a bioluminescent mouse model expressing aromatase PII-promoter-controlled luciferase as a tool for the study of endocrine disrupting chemicals

    SciTech Connect

    Rivest, Patricia Devine, Patrick J. Sanderson, J. Thomas

    2010-11-15

    Dysfunction of the enzyme aromatase (CYP19) is associated with endocrine pathologies such as osteoporosis, impaired fertility and development of hormone-dependent cancers. Certain endocrine disrupting chemicals affect aromatase expression and activity in vitro, but little is known about their ability to do so in vivo. We evaluated a bioluminescent mouse model (LPTA (registered)) CD-1-Tg(Cyp19-luc)-Xen) expressing luciferase under control of the gonadal aromatase pII promoter as an in vivo screening tool for chemicals that may affect aromatase expression. We studied the effects of forskolin, pregnant mare serum gonadotropin and atrazine in this model (atrazine was previously shown to induced pII-promoter-driven aromatase expression in H295R human adrenocortical carcinoma cells). About 2-4 out of every group of 10 male or female Cyp19-luc mice injected i.p. with 10 mg/kg forskolin had increased gonadal bioluminescence after 3-5 days compared to controls; the others appeared non-responsive. Similarly, about 4 per group of 9 individual females injected with pregnant mare serum gonadotropin had increased ovarian bioluminescence after 24 h. There was a statistically significant correlation between ovarian bioluminescence and plasma estradiol concentrations (n = 14; p = 0.022). Males exposed to a single dose of 100 mg/kg or males and females exposed to 5 daily injections of 30 mg/kg atrazine showed no change in gonadal bioluminescence over a 7 day period, but a significant interaction was found between atrazine (100 mg/kg) and time in female mice (p < 0.05; two-way ANOVA). Ex vivo luciferase activity in dissected organs was increased by forskolin in testis, epididymis and ovaries. Atrazine (30 mg/kg/day) increased (30%) luciferase activity significantly in epididymis only. In conclusion, certain individual Cyp19-luc mice are highly responsive to aromatase inducers, suggesting this model, with further optimization, may have potential as an in vivo screening tool for

  8. RILEM TC ISR Summer 2015 Activity Report

    SciTech Connect

    Le Pape, Yann

    2015-08-01

    With aging infrastructures, instances of Alkali Silica Reaction (ASR) and Delayed Ettringite Formation (DEF), broadly covered under the term Internal Swelling Reaction (ISR), are increasingly being detected. They have been observed in bridges, dams, and most recently in nuclear power plants. Concrete swelling may result in bridge partial failure, dams with structural cracks and misaligned turbine shafts, and locked slice gates. For nuclear reactors micro-cracks may cause increased gas permeability which will jeopardize the containment integrity and may decrease the residual structural resistance under accidental loading. This TC, which limits its activity to structures with known expansive concrete, seeks to address two complementary but fundamental questions: a) What is the kinetics of the reaction and b) How would it affect the integrity of the structure (serviceability and strength) and thus establish a science based prognostic to the structure owner.

  9. Luminescence as a Continuous Real-Time Reporter of Promoter Activity in Yeast Undergoing Respiratory Oscillations or Cell Division Rhythms

    PubMed Central

    Robertson, J. Brian; Johnson, Carl Hirschie

    2012-01-01

    This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces cerevisiae that has been genetically modified to express firefly luciferase under the control of a promoter of interest and a continuous culture bioreactor that incorporates a photomultiplier apparatus for detecting light emission. Additionally, this chapter describes a method for observing rhythmic (cell cycle-related) promoter activity in small batch cultures of yeast through luminescence monitoring. PMID:21468985

  10. Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress

    PubMed Central

    Santos, Efrén; Remy, Serge; Thiry, Els; Windelinckx, Saskia; Swennen, Rony; Sági, László

    2009-01-01

    Background Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc+) gene and low temperature-responsive luciferase activation was monitored in real time. Results Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26°C followed by a gradual decrease to 8°C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned via PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the uidA reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed. Conclusion This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout in vitro development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate promoters even in a multicopy T

  11. Advanced Light Source: Activity report 1993

    SciTech Connect

    Not Available

    1994-11-01

    The Advanced Light Source (ALS) produces the world`s brightest light in the ultraviolet and soft x-ray regions of the spectrum. The first low-energy third-generation synchrotron source in the world, the ALS provides unprecedented opportunities for research in science and technology not possible anywhere else. This year marked the beginning of operations and the start of the user research program at the ALS, which has already produced numerous high quality results. A national user facility located at Lawrence Berkeley Laboratory of the University of California, the ALS is available to researchers from academia, industry, and government laboratories. This report contains the following: (1) director`s message; (2) operations overview; (3) user program; (4) users` executive committee; (5) industrial outreach; (6) accelerator operations; (7) beamline control system; (8) insertion devices; (9) experimental systems; (10) beamline engineering; (11) first results from user beamlines; (12) beamlines for 1994--1995; (13) special events; (14) publications; (15) advisory panels; and (16) ALS staff.

  12. Severe Accident Test Station Activity Report

    SciTech Connect

    Pint, Bruce A.; Terrani, Kurt A.

    2015-06-01

    Enhancing safety margins in light water reactor (LWR) severe accidents is currently the focus of a number of international R&D programs. The current UO2/Zr-based alloy fuel system is particularly susceptible since the Zr-based cladding experiences rapid oxidation kinetics in steam at elevated temperatures. Therefore, alternative cladding materials that offer slower oxidation kinetics and a smaller enthalpy of oxidation can significantly reduce the rate of heat and hydrogen generation in the core during a coolant-limited severe accident. In the U.S. program, the high temperature steam oxidation performance of accident tolerant fuel (ATF) cladding solutions has been evaluated in the Severe Accident Test Station (SATS) at Oak Ridge National Laboratory (ORNL) since 2012. This report summarizes the capabilities of the SATS and provides an overview of the oxidation kinetics of several candidate cladding materials. A suggested baseline for evaluating ATF candidates is a two order of magnitude reduction in the steam oxidation resistance above 1000ºC compared to Zr-based alloys. The ATF candidates are categorized based on the protective external oxide or scale that forms during exposure to steam at high temperature: chromia, alumina, and silica. Comparisons are made to literature and SATS data for Zr-based alloys and other less-protective materials.

  13. Novel Stably Transfected Human Reporter Cell Line AIZ-AR as a Tool for an Assessment of Human Androgen Receptor Transcriptional Activity

    PubMed Central

    Bartonkova, Iveta; Novotna, Aneta; Dvorak, Zdenek

    2015-01-01

    Androgen receptor plays multiple physiological and pathological roles in human organism. In the current paper, we describe construction and characterization of a novel stably transfected human reporter cell line AIZ-AR for assessment of transcriptional activity of human androgen receptor. Cell line AIZ-AR is derived from human prostate carcinoma epithelial cell line 22Rv1 that was transfected with reporter plasmid containing 3 copies of androgen response regions (ARRs) followed by a single copy of androgen response element (ARE) from the promoter region of human prostate specific antigen (PSA) gene. AIZ-AR cells remained fully functional for more than 60 days and over 25 passages in the culture and even after cryopreservation. Time-course analyses showed that AIZ-AR cells allow detection of AR ligands as soon as after 8 hours of the treatment. We performed dose-response analyses with 23 steroids in 96-well plate format. We observed activation of AR by androgens, but not by estrogens and mineralocorticoids. Some glucocorticoids and progesterone also induced luciferase, but their potencies were 2-3 orders of magnitude weaker as compared to androgens. Taken together, we have developed a rapid, sensitive, selective, high-throughput and reproducible tool for detection of human AR ligands, with potential use in pharmacological and environmental applications. PMID:25811655

  14. Warm Water Bath Stimulates Phase-Shifts of the Peripheral Circadian Clocks in PER2::LUCIFERASE Mouse

    PubMed Central

    Kuriki, Daisuke; Haraguchi, Atsushi; Shibata, Shigenobu

    2014-01-01

    Circadian clocks in the peripheral tissues of mice are known to be entrained by pulse stimuli such as restricted feeding, novel wheel running, and several other agents. However, there are no reports on high temperature pulse-mediated entrainment on the phase-shift of peripheral clocks in vivo. Here we show that temperature treatment of mice for two days at 41°C, instead of 37°C, for 1–2 h during the inactive period, using a temperature controlled water bath stimulated phase-advance of peripheral clocks in the kidney, liver, and submandibular gland of PER2::LUCIFERASE mice. On the other hand, treatment for 2 days at 35°C ambient room temperature for 2 h did not cause a phase-advance. Maintenance of mice at 41°C in a water bath, sustained the core body temperature at 40–41°C. However, the use of 37°C water bath or the 35°C ambient room temperature elevated the core body temperature to 38.5°C, suggesting that at least a core body temperature of 40–41°C is necessary to cause phase-advance under light-dark cycle conditions. The temperature pulse stimulation at 41°C, instead of 37°C water bath for 2 h led to the elevated expression of Per1 and Hsp70 in the peripheral tissue of mice. In summary, the present study demonstrates that transient high temperature pulse using water bath during daytime causes phase-advance in mouse peripheral clocks in vivo. The present results suggest that hot water bath may affect the phase of peripheral clocks. PMID:24933288

  15. General aviation activity survey. Annual summary report for 1992

    SciTech Connect

    Not Available

    1992-01-01

    This report presents the results of the annual General Aviation Activity Survey. The survey is conducted by the FAA to obtain information on the flight activity of the United States registered general aviation aircraft fleet. The report contains breakdowns of active aircraft, annual flight hours, average flight hours and other statistics by manufacturer/model group, aircraft type, state and region of based aircraft, and primary use. Also included are fuel consumption, lifetime airframe hours, engine hours, miles flown estimates, estimates of the number of landings, IFR hours flown, and grade of fuel consumed by the general aviation fleet. Aircraft, Aircraft activity, Aircraft use, Fuel consumption, General aviation, Hours flown, Miles flown.

  16. GENERATION OF TWO NOVEL CELL LINES THAT STABLY EXPRESS HAR AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Generation of Two Novel Cell Lines that Stably Express hAR and Firefly Luciferase Genes for Endocrine Screening
    K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1
    1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reproductive Toxicology Divi...

  17. Sol-gel immobilized luciferase-based ATP biosensor for meat quality determination in postmortem pig muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inferior pork quality results from a rapid rate of muscle metabolism that is stimulated by ATP depletion early postmortem. The objectives of this study were 1) to test the hypothesis that muscle ATP as measured by a luciferase assay could be used for predicting fresh pork quality, and 2) to explore ...

  18. The rapid quantitation of the filamentous blue-green alga plectonema boryanum by the luciferase assay for ATP

    NASA Technical Reports Server (NTRS)

    Bush, V. N.

    1974-01-01

    Plectonema boryanum is a filamentous blue green alga. Blue green algae have a procaryotic cellular organization similar to bacteria, but are usually obligate photoautotrophs, obtaining their carbon and energy from photosynthetic mechanism similar to higher plants. This research deals with a comparison of three methods of quantitating filamentous populations: microscopic cell counts, the luciferase assay for ATP and optical density measurements.

  19. NASA's Microgravity Technology Report: Summary of Activities 1997

    NASA Technical Reports Server (NTRS)

    Woodard, Dan

    1998-01-01

    The purpose of the 1997 NASA Microgravity Technology Report is to update the Microgravity Research Program's technology development policy and to present and assess current technology related activities and requirements identified within its research and technology disciplines.

  20. 12 CFR 1282.63 - Annual Housing Activities Report.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 9 2012-01-01 2012-01-01 false Annual Housing Activities Report. 1282.63 Section 1282.63 Banks and Banking FEDERAL HOUSING FINANCE AGENCY HOUSING GOALS AND MISSION ENTERPRISE... Acts. Each Enterprise shall submit such report within 75 days after the end of each calendar year,...

  1. A Self-Report Measure of Physical Activity

    ERIC Educational Resources Information Center

    Siegel, Donald

    2005-01-01

    There are multiple approaches to measuring physical activity. Among these are direct observation, electronic monitoring, direct and indirect calorimetry, and self-report instruments. Self-report instruments are the most practical and cost effective option for use with a large group. In a study by Motl, Dishman, Dowda, and Pate (2004), two groups…

  2. 24 CFR 1710.310 - Annual report of activity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Annual report of activity. 1710.310 Section 1710.310 Housing and Urban Development Regulations Relating to Housing and Urban Development... URBAN DEVELOPMENT (INTERSTATE LAND SALES REGISTRATION PROGRAM) LAND REGISTRATION Reporting...

  3. 11 CFR 300.36 - Reporting Federal election activity; recordkeeping.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 11 Federal Elections 1 2011-01-01 2011-01-01 false Reporting Federal election activity; recordkeeping. 300.36 Section 300.36 Federal Elections FEDERAL ELECTION COMMISSION BIPARTISAN CAMPAIGN REFORM ACT OF 2002-(BCRA) REGULATIONS NON-FEDERAL FUNDS State, District, and Local Party Committees and Organizations § 300.36 Reporting Federal...

  4. Advanced Light Source Activity Report 1997/1998

    SciTech Connect

    Greiner, Annette

    1999-03-01

    This Lawrence Berkeley National Laboratory, Advanced Light Source (ALS) activity report for 1997/98 discusses the following topics: Introduction and Overview; Science Highlights; Facility Report; Special Events; ALS Advisory Panels 1997/98; ALS Staff 1997/98 and Facts and Figures for the year.

  5. Aeronautics and Space Report of the President: 1975 Activities.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    This report, submitted to the Congress by President Ford in accordance with the National Aeronautics and Space Act of 1958, summarizes the United States' space and aeronautics activities for the year 1975. Detailed summaries of the activities of the following governmental departments or agencies are provided: National Aeronautics and Space…

  6. Report of National Collegiate Alcohol Awareness Week Activities Fall 1987.

    ERIC Educational Resources Information Center

    Owens, Ann English; And Others

    This document presents a report of the education and prevention activities recognizing National Collegiate Alcohol Awareness (NCAA) Week undertaken at Central Michigan University in Mt. Pleasant, Michigan during October and early November, 1987. It begins with a brief review of the university's campus-wide programs, goals, and activities to reduce…

  7. Worksite Health Promotion Activities. 1992 National Survey. Summary Report.

    ERIC Educational Resources Information Center

    Public Health Service (DHHS), Rockville, MD. Office of Disease Prevention and Health Promotion.

    The survey reported in this document examined worksite health promotion and disease prevention activities in 1,507 private worksites in the United States. Specificlly, the survey assessed policies, practices, services, facilities, information, and activities sponsored by employers to improve the health of their employees, and assessed health…

  8. Thermodynamics of anesthetic/protein interactions. Temperature studies on firefly luciferase.

    PubMed Central

    Dickinson, R; Franks, N P; Lieb, W R

    1993-01-01

    Firefly luciferase is a soluble enzyme which is unusually sensitive to general anesthetics. The inhibition of the highly purified enzyme by three inhalational and three alcohol general anesthetics has been studied as a function of temperature, in the range from 5 to 20 degrees C. Inhibition constants Ki were determined at different temperatures, and van't Hoff plots of ln (Ki) versus reciprocal absolute temperature were found to be linear for all agents. Analysis of these plots gave values for the standard Gibbs free energy, enthalpy and entropy changes for transferring each anesthetic from water to the anesthetic-binding pocket on the protein. The most striking finding was that the enthalpy changes were much more negative for anesthetics binding to the protein than for binding to lipids or simple solvents. Furthermore, amongst the set of anesthetics studied, it was found that increasing potency correlated with favorable enthalpy rather than entropy changes. We discuss our results with respect to the molecular mechanisms underlying general anesthesia. PMID:8494981

  9. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    NASA Technical Reports Server (NTRS)

    Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

    1977-01-01

    The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

  10. Two-stage prediction of the effects of imidazolium and pyridinium ionic liquid mixtures on luciferase.

    PubMed

    Ge, Hui-Lin; Liu, Shu-Shen; Su, Bing-Xia; Zhu, Xiang-Wei

    2014-01-01

    The predicted toxicity of mixtures of imidazolium and pyridinium ionic liquids (ILs) in the ratios of their EC50, EC10, and NOEC (no observed effect concentration) were compared to the observed toxicity of these mixtures on luciferase. The toxicities of EC50 ratio mixture can be effectively predicted by two-stage prediction (TSP) method, but were overestimated by the concentration addition (CA) model and underestimated by the independent action (IA) model. The toxicities of EC10 ratio mixtures can be basically predicted by TSP and CA, but were underestimated by IA. The toxicities of NOEC ratio mixtures can be predicted by TSP and CA in a certain concentration range, but were underestimated by IA. Our results support the use of TSP as a default approach for predicting the combined effect of different types of ILs at the molecular level. In addition, mixtures of ILs mixed at NOEC and EC10 could cause significant effects of 64.1% and 97.7%, respectively. Therefore, we should pay high attention to the combined effects in mixture risk assessment. PMID:24858273

  11. Internet Reporting of Weekly Physical Activity Behaviors: The WIN Study

    PubMed Central

    Bain, Tyson; Frierson, Georita M.; Trudelle-Jackson, Elaine; Morrow, James R.

    2009-01-01

    Background Self-report measures have been validated and are widely used. Interest currently lies in the development of simple, valid methods that can be used in any location to determine level of PA in large populations/samples. The purpose of this report is to illustrate tracking of physical activity behaviors and musculoskeletal injury reports on a weekly basis via the Internet. Methods The Women’s Injury Study (WIN) methodology includes use of BRFSS-related physical activity items that are completed online by more than 800 women weekly for an average of 3 years. Results With more than 45,000 weekly physical activity and injury logs, the percentage of total logs submitted via online records is 91%. Self-reported pedometer steps are consistent with similar, smaller research samples. Conclusions This report suggests that Internet tracking is a viable means of assessing nearly real-time physical activity, describes the process of developing and monitoring self-reported physical activity behaviors via the Internet, and provides recommendations for others considering such methods. PMID:20683095

  12. Disruption of bbe02 by Insertion of a Luciferase Gene Increases Transformation Efficiency of Borrelia burgdorferi and Allows Live Imaging in Lyme Disease Susceptible C3H Mice

    PubMed Central

    Chan, Kamfai; Alter, Laura; Barthold, Stephen W.; Parveen, Nikhat

    2015-01-01

    Lyme disease is the most prevalent tick-borne disease in North America and Europe. The causative agent, Borrelia burgdorferi persists in the white-footed mouse. Infection with B. burgdorferi can cause acute to persistent multisystemic Lyme disease in humans. Some disease manifestations are also exhibited in the mouse model of Lyme disease. Genetic manipulation of B. burgdorferi remains difficult. First, B. burgdorferi contains a large number of endogenous plasmids with unique sequences encoding unknown functions. The presence of these plasmids needs to be confirmed after each genetic manipulation. Second, the restriction modification defense systems, including that encoded by bbe02 gene lead to low transformation efficiency in B. burgdorferi. Therefore, studying the molecular basis of Lyme pathogenesis is a challenge. Furthermore, investigation of the role of a specific B. burgdorferi protein throughout infection requires a large number of mice, making it labor intensive and expensive. To overcome the problems associated with low transformation efficiency and to reduce the number of mice needed for experiments, we disrupted the bbe02 gene of a highly infectious and pathogenic B. burgdorferi strain, N40 D10/E9 through insertion of a firefly luciferase gene. The bbe02 mutant shows higher transformation efficiency and maintains luciferase activity throughout infection as detected by live imaging of mice. Infectivity and pathogenesis of this mutant were comparable to the wild-type N40 strain. This mutant will serve as an ideal parental strain to examine the roles of various B. burgdorferi proteins in Lyme pathogenesis in the mouse model in the future. PMID:26069970

  13. Metastasizing, Luciferase Transduced MAT-Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug

    PubMed Central

    Jantscheff, Peter; Esser, Norbert; Geipel, Andreas; Woias, Peter; Ziroli, Vittorio; Goldschmidtboing, Frank; Massing, Ulrich

    2011-01-01

    The most fatal outcomes of prostate carcinoma (PCa) result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24), spleen (3/24), kidney (4/24), liver (5/24), and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively). Preliminary data of orthotopic implantation (three animals) showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes). Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4), lung (3/6) or lumbar spine (0/2), as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s) to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented. PMID:24212827

  14. NSLS 2005 ACTIVITY REPORT (NATIONAL SYNCHROTRON LIGHT SOURCE ACTIVITY REPORT 2005).

    SciTech Connect

    MILLER, L.

    2006-05-01

    In 2005, the NSLS proved itself, once again, to be a center of scientific excellence. This remarkable facility, commissioned in the early 1980s, is still attracting some of the world's best researchers in almost every scientific field, who produce more than seven hundred scientific papers every year using the NSLS. The 'Science Highlights' and 'Feature Highlights' sections of this report are just a small sampling of the many, many impressive research projects conducted at the NSLS in 2005. For example, a user group synthesized and studied zinc-oxide nanowires, which have applications in many optical and electrical devices. Another user group studied how strontium and uranium are removed from high-level radioactive waste. And in another interesting study, users deciphered the basis for antibiotic resistance. However, as always, the success of these projects depends on the performance of the facility. Again this year, the rings were in top form--reliability was 96 percent for the x-ray ring and 99 percent for the VUV-IR ring. Additionally, to keep the NSLS as productive as possible and to continue to attract users, many beamline upgrade projects were completed this year. One of the highlights of these upgrades is the new mini-gap undulator installed at beamline X25. This insertion device is providing a much brighter x-ray source for the program at X25. In the always important area of safety, several noteworthy activities took place this year. In particular, NSLS staff made a major commitment to labeling and inspecting electrical equipment. And perhaps the best news is what didn't happen--there were no reportable occurrences related to environmental, safety, or health issues in 2005, and no injuries that resulted in restricted or lost time. We all owe thanks to the dedicated NSLS staff and users who have ensured that the NSLS remains a reliable, safe, up-to-date research facility. As 2005 came to an end, I stepped down as NSLS Chairman in order to focus my primary

  15. Towards a new paradigm: Activity level balanced sustainability reporting.

    PubMed

    Samudhram, Ananda; Siew, Eu-Gene; Sinnakkannu, Jothee; Yeow, Paul H P

    2016-11-01

    Technoeconomic paradigms based economic growth theories suggest that waves of technological innovations drove the economic growth of advanced economies. Widespread economic degradation and pollution is an unintended consequence of such growth. Tackling environmental and social issues at firm levels would help us to overcome such issues at macro-levels. Consequently, the Triple Bottom Line (TBL) reporting approach promotes firm level economic, environmental and social performances. Incorporating Zink's (2014) 3-pillar presentation model, this paper indicates that economic, social and environmental performances tend to be reported at firm level. All three pillars are not covered evenly at the activity levels. Thus, a loophole is identified whereby excellent environmental performance at activity levels could potentially leave poor social performance undisclosed. A refinement of the TBL paradigm, whereby all three pillars are covered at the activity level, is suggested, to enhance sustainability reporting. PMID:27029522

  16. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). Methods VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Results Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. Conclusions A VRP based CHIKV neutralization assay using Gluc as readout

  17. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  18. NSLS 2007 Activity Report (National Synchrotron Light Source Activity Report 2007)

    SciTech Connect

    Miller ,L.; Nasta, K.

    2008-05-01

    The National Synchrotron Light Source is one of the world's most productive and cost-effective user facilities. With 2,219 individual users, about 100 more than last year, and a record-high 985 publications, 2007 was no exception. In addition to producing an impressive array of science highlights, which are included in this Activity Report, many NSLS users were honored this year for their scientific accomplishments. Throughout the year, there were major strides in the development of the scientific programs by strengthening strategic partnerships with major research resources and with the Center for Functional Nanomaterials (CFN). Of particular note, the Consortium for Materials Properties Research in Earth Sciences (COMPRES) received renewed funding for the next five years through the National Science Foundation. COMPRES operates four high-pressure NSLS beamlines--X17B2, X17B3, X17C, and U2A--and serves the earth science community as well as the rapidly expanding segment of researchers using high-pressure techniques in materials, chemical, and energy-related sciences. A joint appointment was made between the NSLS and Stony Brook University to further enhance interactions with COMPRES. There was major progress on two key beamline projects outlined in the Five-Year Strategic Plan: the X25 beamline upgrade and the construction of the X9 small angle scattering (SAXS) beamline. The X25 overhaul, which began with the installation of the in-vacuum mini-gap undulator (MGU) in January 2006, is now complete. X25 is once again the brightest beamline for macromolecular crystallography at the NSLS, and in tandem with the X29 undulator beamline, it will keep the NSLS at the cutting edge in this important area of research. Upgrade work associated with the new MGU and the front end for the X9 SAXS beamline--jointly developed by the NSLS and the CFN--also was completed. Beamline X9 will host the SAXS program that currently exists at beamline X21 and will provide new microbeam SAXS

  19. Two techniques for eliminating luminol interference material and flow system configurations for luminol and firefly luciferase systems

    NASA Technical Reports Server (NTRS)

    Thomas, R. R.

    1976-01-01

    Two methods for eliminating luminol interference materials are described. One method eliminates interference from organic material by pre-reacting a sample with dilute hydrogen peroxide. The reaction rate resolution method for eliminating inorganic forms of interference is also described. The combination of the two methods makes the luminol system more specific for bacteria. Flow system designs for both the firefly luciferase and luminol bacteria detection systems are described. The firefly luciferase flow system incorporating nitric acid extraction and optimal dilutions has a functional sensitivity of 3 x 100,000 E. coli/ml. The luminol flow system incorporates the hydrogen peroxide pretreatment and the reaction rate resolution techniques for eliminating interference. The functional sensitivity of the luminol flow system is 1 x 10,000 E. coli/ml.

  20. Dehydroluciferyl-AMP is the main intermediate in the luciferin dependent synthesis of Ap4A catalyzed by firefly luciferase.

    PubMed

    Fontes, R; Ortiz, B; de Diego, A; Sillero, A; Günther Sillero, M A

    1998-11-01

    It was previously assumed that E x LH2-AMP was the intermediate complex in the synthesis of Ap4A catalyzed by firefly luciferase (EC 1.13.12.7), when luciferin (LH2) was used as cofactor. However, here we show that LH2 is partly transformed, shortly after the onset of the luciferase reaction, to dehydroluciferin (L) with formation of an E x L-AMP complex which is the main intermediate for the synthesis of Ap4A. Formation of three more derivatives of LH2 were also observed, related to the production of light by the enzyme. CoA, a known stimulator of light production, inhibits the synthesis of Ap4A by reacting with the E x L-AMP complex and yielding L-CoA. PMID:9827543

  1. 1994 Activity Report, National Synchrotron Light Source. Annual report, October 1, 1993-September 30, 1994

    SciTech Connect

    Rothman, E.Z.

    1995-05-01

    This report is a summary of activities carried out at the National Synchrotron Light Source during 1994. It consists of sections which summarize the work carried out in differing scientific disciplines, meetings and workshops, operations experience of the facility, projects undertaken for upgrades, administrative reports, and collections of abstracts and publications generated from work done at the facility.

  2. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  3. An adenoviral vector for probing promoter activity in primary immune cells

    PubMed Central

    Tripathi, Pulak; Madan, Rajat; Chougnet, Claire; Divanovic, Senad; Ma, Xiaojing; Wahl, Larry M.; Gajewski, Thomas; Karp, Christopher L.; Hildeman, David A.

    2010-01-01

    Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter–reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells. PMID:16563424

  4. A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren

    PubMed Central

    Igarashi, Masayuki; Doe, Matsumi; Tamaru, Aki; Kinoshita, Naoko; Ogura, Yoshitoshi; Iwamoto, Tomotada; Sawa, Ryuichi; Umekita, Maya; Enany, Shymaa; Nishiuchi, Yukiko; Osada-Oka, Mayuko; Hayashi, Tetsuya; Niki, Mamiko; Tateishi, Yoshitaka; Hatano, Masaki

    2015-01-01

    Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904–1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0–7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904–1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904–1. Our method provides a

  5. TGEV infection up-regulates FcRn expression via activation of NF-κB signaling

    PubMed Central

    Guo, Jinyue; Li, Fei; Qian, Shaoju; Bi, Dingren; He, Qigai; Jin, Hui; Luo, Rui; Li, Shaowen; Meng, Xianrong; Li, Zili

    2016-01-01

    It has been well characterized that the neonatal Fc receptor (FcRn) transports maternal IgG to a fetus or newborn and protects IgG from degradation. We previously reported that FcRn is expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2). Transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (TGEV). How porcine FcRn (pFcRn) expression is regulated by pathogenic infection remains unknown. Our research shows that IPEC-J2 cells infected with TGEV had up-regulated pFcRn expression. In addition, the NF-κB signaling pathway was activated in IPEC-J2 cells by TGEV infection. Furthermore, treatment of TGEV-infected IPEC-J2 cells with the NF-κB-specific inhibitor BAY 11-7082 resulted in down-regulation of pFcRn expression. Transient transfection of pFcRn promoter luciferase report plasmids with overexpression of NF-κB p65 transcription factor enhanced the activation of the luciferase report plasmids. We identified four NF-κB transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. Together, the data provide the first evidence that TGEV infection up-regulates pFcRn expression via activation of NF-κB signaling. PMID:27555521

  6. TGEV infection up-regulates FcRn expression via activation of NF-κB signaling.

    PubMed

    Guo, Jinyue; Li, Fei; Qian, Shaoju; Bi, Dingren; He, Qigai; Jin, Hui; Luo, Rui; Li, Shaowen; Meng, Xianrong; Li, Zili

    2016-01-01

    It has been well characterized that the neonatal Fc receptor (FcRn) transports maternal IgG to a fetus or newborn and protects IgG from degradation. We previously reported that FcRn is expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2). Transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (TGEV). How porcine FcRn (pFcRn) expression is regulated by pathogenic infection remains unknown. Our research shows that IPEC-J2 cells infected with TGEV had up-regulated pFcRn expression. In addition, the NF-κB signaling pathway was activated in IPEC-J2 cells by TGEV infection. Furthermore, treatment of TGEV-infected IPEC-J2 cells with the NF-κB-specific inhibitor BAY 11-7082 resulted in down-regulation of pFcRn expression. Transient transfection of pFcRn promoter luciferase report plasmids with overexpression of NF-κB p65 transcription factor enhanced the activation of the luciferase report plasmids. We identified four NF-κB transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. Together, the data provide the first evidence that TGEV infection up-regulates pFcRn expression via activation of NF-κB signaling. PMID:27555521

  7. Performing Clerical Activities. Preparing Accident Reports in an Office.

    ERIC Educational Resources Information Center

    Jones, Maleeta M.

    Supporting performance objective 5 of the V-TECS (Vocational-Technical Education Consortium of States) Secretarial Catalog, both a set of student materials and an instructor's manual on preparing accident reports in an office are included in this packet. (The packet is the third in a set of three on performing clerical activities--CE 016 948-950.)…

  8. Pupil Inquiry Behavior Analysis and Change Activity. Interim Project Report.

    ERIC Educational Resources Information Center

    Manion, Raymond C.

    This interim report discusses progress toward three major goals of the Pupil Inquiry Behavior Analysis and Change Activity: increased pupil inquiry, changed teacher behavior to facilitate pupil inquiry, and the development of a 32-week course of instruction to provide for these behavioral changes. Data currently available deals with the emotional…

  9. Mathemagenic Activities Program: [Reports on Cognitive/Language Development].

    ERIC Educational Resources Information Center

    Smock, Charles D., Ed.

    This set of 13 research reports, bulletins and papers is a product of the Mathemagenic Activities Program (MAP) for early childhood education of the University of Georgia Follow Through Program. Based on Piagetian theory, the MAP provides sequentially structured sets of curriculum materials and processes that are designed to continually challenge…

  10. 10 CFR 490.507 - Credit activity reporting requirements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 490.507 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ALTERNATIVE FUEL TRANSPORTATION PROGRAM Alternative Fueled Vehicle Credit Program § 490.507 Credit activity reporting requirements. (a) A covered person or fleet applying for allocation of alternative fueled vehicle credits must submit a...

  11. 10 CFR 490.507 - Credit activity reporting requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 490.507 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ALTERNATIVE FUEL TRANSPORTATION PROGRAM Alternative Fueled Vehicle Credit Program § 490.507 Credit activity reporting requirements. (a) A covered person or fleet applying for allocation of alternative fueled vehicle credits must submit a...

  12. Report of the International Bureau of Education on Its Activities.

    ERIC Educational Resources Information Center

    United Nations Educational, Scientific, and Cultural Organization, Paris (France). General Conference.

    This second report by the Council of the International Bureau of Education covers the period since the close of the sixteenth session of the General Conference to 31 July 1972. It deals with the activities of the Council itself, the steps taken to develop the programme of the International Bureau of Education during the period under review, and…

  13. Section on Cataloguing: Report of the Activities, 2000-2001.

    ERIC Educational Resources Information Center

    Witt, Maria

    This paper reports on the activities of the IFLA (International Federation of Library Associations and Institutions) Section on Cataloging for 2000-2001. The first part discusses the interest in cataloging and its harmonization at the international level that is shared by libraries throughout the world, including the section's work on development…

  14. Annual Report for 2003 Wild Horse Research and Field Activities

    USGS Publications Warehouse

    Ransom, Jason; Singer, Francis J.; Zeigenfuss, Linda C.

    2004-01-01

    This report is meant to highlight the activities of the 2003 field season, as well as to provide a general overview of the data collected. More in-depth data analysis will be conducted following the conclusion of each I phase of the research project, and in many cases will not be possible until several seasons of data are collected.

  15. Report on active and planned spacecraft and experiments

    NASA Technical Reports Server (NTRS)

    Vette, J. I. (Editor); Vostreys, R. W. (Editor)

    1977-01-01

    Information concerning active and planned spacecraft and experiments is reported. The information includes a wide range of disciplines: astronomy, earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. These spacecraft projects represent the efforts and funding of individual countries as well as cooperative arrangements among different countries.

  16. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  17. Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays.

    PubMed

    Klopčič, Ivana; Skledar, Darja Gramec; Mašič, Lucija Peterlin; Dolenc, Marija Sollner

    2016-10-01

    The anti-androgenic and anti-thyroid hormonal activities of the two novel brominated flame retardants, TBB and TBPH and of their metabolites TBBA and TBMEPH have been compared using the luciferase reporter gene assays. Only the parent compounds TBB and TBPH exhibited anti-glucocorticoid activity with IC50 values of 1.9 μM and 0.3 μM. Furthermore, mode of action for these two compounds is by direct competing to the glucocorticoid receptor (GR) with IC50 values of 0.03 μM and 0.002 μM. All four tested compounds possess anti-androgenic and anti-thyroid hormonal activities, without agonist activities on the respective receptors. Anti-androgenic activities with IC50 values of 43.5 μM, 0.1 μM, 47.5 μM and 1.3 μM were found for TBB, TBPH, TBBA and TBMEPH. The anti-thyroid hormonal IC50 values of 37.5 μM, 0.1 μM, 22.8 μM and 32.3 μM for TBB, TBPH, TBBA and TBMEPH, together with the above quoted results, indicate that metabolism can modify anti-androgenic, anti-glucocorticoid and anti-thyroid hormonal effects of these novel brominated flame retardants. Furthermore, the parent flame retardants are shown to be able to disrupt the function of the GR as antagonists by direct competition to the receptor. PMID:27380226

  18. NASA's Microgravity Technology Report, 1996: Summary of Activities

    NASA Technical Reports Server (NTRS)

    Kierk, Isabella

    1996-01-01

    This report covers technology development and technology transfer activities within the Microgravity Science Research Programs during FY 1996. It also describes the recent major tasks under the Advanced Technology Development (ATD) Program and identifies current technology requirements. This document is consistent with NASA,s Enteprise for the Human Exploration and development of Space (HEDS) Strategic Plan. This annual update reflects changes in the Microgravity Science Research Program's new technology activities and requirements. Appendix A. FY 1996 Advanced Technology Development. Program and Project Descriptions. Appendix B. Technology Development.

  19. Final Report: Imaging of Buried Nanoscale Optically Active Materials

    SciTech Connect

    Appelbaum, Ian

    2011-07-05

    This is a final report covering work done at University of Maryland to develop a Ballistic Electron Emission Luminescence (BEEL) microscope. This technique was intended to examine the carrier transport and photon emission in deeply buried optically-active layers and thereby provide a means for materials science to unmask the detailed consequences of experimentally controllable growth parameters, such as quantum dot size, statistics and orientation, and defect density and charge recombination pathways.

  20. Geomagnetic activity and Hale sector boundaries. Technical report

    SciTech Connect

    Lundstedt, H.; Scherrer, P.H.; Wilcox, J.M.

    1980-02-01

    The variation of the geomagnetic activity index Ap at the Interplanetary Magnetic Field (IMF) sector boundaries (+ to - and - to +) has been studied for three solar cycles, separating data into vernal and autumnal equinoxes. It was found that a reported increase in Ap as an effect of a Hale boundary can be better attributed to the occurrence of a negative IMF Bz component in the geocentric solar magnetospheric coordinate system and to the occurrence of high speed solar wind streams.

  1. Activity-based costing for electric utilities. Final report

    SciTech Connect

    Croyle, D.R.; Schapiro, I.A.; Keglevic, P.M.

    1992-08-01

    This EPRI report is a ``primer`` on Activity-Based Costing (ABC). ABC is a cost management aproach which can make an important contribution to understanding and controlling the changing costs in the electric utility industry. It is a method for attributing costs to activities, products and services by better understanding the underlying factors which drive those costs. ABC can help utility managers make better decisions through the application of more accurate process and product cost information and a fuller understanding of which activities add value and which do not. Armed with such information, utility managers are better equipped to address many of the strategic and operating decisions which they routinely face. The report introduces the ABC concept and approach to utility managers and offers insights into how ABC can be and is being used to control costs and improve strategic and operating decisions in electric utilities and other industries. The report (1) describes the ABC approach, (2) discusses the value of ABC to elecuic utilities, (3) identifies potential applications of ABC to current utility issues, (4) describes a step-by-step approach to developing and implementing ABC in the utility environment, and (5) presents a survey of more than 30 electric utilities and several detailed case studies of electric utilities and other companies who have adopted and are using ABC.

  2. Triennial Report 2006-2009. Commission 10: Solar Activity

    NASA Technical Reports Server (NTRS)

    Klimchuk, James A.

    2008-01-01

    Commission 10 deals with solar activity in all of its forms, ranging from the smallest nanoflares to the largest coronal mass ejections. This report reviews scientific progress over the roughly two-year period ending in the middle of 2008. This has been an exciting time in solar physics, highlighted by the launches of the Hinode and STEREO missions late in 2006. The report is reasonably comprehensive, though it is far from exhaustive. Limited space prevents the inclusion of many significant results. The report is divided into following sections: Photosphere and Chromosphere; Transition Region; Corona and Coronal Heating; Coronal Jets; Flares; Coronal Mass Ejection Initiation; Global Coronal Waves and Shocks; Coronal Dimming; The Link Between Low Coronal CME signatures and Magnetic Clouds; Coronal Mass Ejections in the Heliosphere; and Coronal Mass Ejections and Space Weather. Primary authorship is indicated at the beginning of each section.

  3. NSLS 2006 ACTIVITY REPORT (NATIONAL SYNCHROTRON LIGHT SOURCE ACTIVITY REPORT 2006)

    SciTech Connect

    MILLER, L.

    2006-12-31

    and the years to follow, we need to continue to educate our elected representatives about the benefits that are provided to our society and our economy by scientific investigation including research done at DOE user facilities like the NSLS. We face another interesting challenge as the NSLS-II project progresses: the formation of scientific research teams associated with particular beamlines at the new facility. In early 2007, the final draft of the conceptual design report will be available, which will describe the projected capabilities of NSLS-II, and we can expect a workshop in mid-2007 to launch the process leading to letters of intent for beamlines. This process will include lots of discussion about access modes, as we seek ways to allow scientific and technical innovators from the user community to play significant roles at NSLS-II.

  4. A Protein-Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases.

    PubMed

    Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi

    2016-01-01

    There is a significant focus on detecting and assaying protein-protein interactions (PPIs) in biology and biotechnology. Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent protein-coding polypeptide, as well as Förster resonance energy transfer (FRET). Here, we describe a novel PPI assay FlimPIA (firefly luminescent intermediate-based protein-protein interaction assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc). PMID:27424900

  5. Determination of sterilization effectiveness by measuring bacterial growth in a biological indicator through firefly luciferase determination of ATP.

    PubMed

    Webster, J J; Walker, B G; Ford, S R; Leach, F R

    1988-01-01

    A bioluminescence procedure for measurement of microbial ATP allows a rapid determination of the effectiveness of autoclave sterilization. This determination is achieved faster than detection of acid production in a biological indicator via a pH indicator. Bacterial outgrowth from spores on test strips of the biological indicator was detected by measurement of ATP using the firefly luciferase reaction. A measureable increase in ATP was found after 5 hours of incubation of a biological indicator that had been treated under sterilizing conditions that produced 75% sterility of the biological indicator as measured by acid production. This is a marked improvement over the 24-48 hours of incubation currently required. PMID:3213598

  6. A novel reporter system for neutralizing and enhancing antibody assay against dengue virus

    PubMed Central

    2014-01-01

    Background Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. Results In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. Conclusions This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies. PMID:24548533

  7. Report on Federal Activities under the Rehabilitation Act. Annual Report, Fiscal Year 2001

    ERIC Educational Resources Information Center

    US Department of Education, 2005

    2005-01-01

    The Rehabilitation Act of 1973, as amended, provides the legislative basis for programs and activities that assist individuals with disabilities in the pursuit of gainful employment, independence, self-sufficiency and full integration into community life. This report is intended to provide a description of accomplishments and progress made under…

  8. Rehabilitation Services Administration Annual Report, Fiscal Year 2004: Report on Federal Activities under the "Rehabilitation Act"

    ERIC Educational Resources Information Center

    US Department of Education, 2007

    2007-01-01

    The "Rehabilitation Act of 1973," as amended (the act), provides the legislative basis for programs and activities that assist individuals with disabilities in the pursuit of gainful employment, independence, self-sufficiency and full integration into community life. This report is intended to provide a description of accomplishments and progress…

  9. 12 CFR 163.180 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... proceeding or in response to a request for disclosure of non-public information under 12 CFR 4.33 or 12 CFR... Suspicious Activity Report. (3) SARs required. A savings association or service corporation shall file a SAR... other institution-affiliated parties to supervisory action. (11) Obtaining SARs. A savings...

  10. 12 CFR 163.180 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... proceeding or in response to a request for disclosure of non-public information under 12 CFR 4.33 or 12 CFR... Suspicious Activity Report. (3) SARs required. A savings association or service corporation shall file a SAR... other institution-affiliated parties to supervisory action. (11) Obtaining SARs. A savings...

  11. 12 CFR 390.355 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Activity Report on the form prescribed by the FDIC. (3) SARs required. A State savings association shall... to supervisory action. (11) Obtaining SARs. A State savings association may obtain SARs and the... SARs. SARs are confidential. Any institution or person subpoenaed or otherwise requested to disclose...

  12. 12 CFR 163.180 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... proceeding or in response to a request for disclosure of non-public information under 12 CFR 4.33 or 12 CFR... Suspicious Activity Report. (3) SARs required. A savings association or service corporation shall file a SAR... other institution-affiliated parties to supervisory action. (11) Obtaining SARs. A savings...

  13. 12 CFR 390.355 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Activity Report on the form prescribed by the FDIC. (3) SARs required. A State savings association shall... to supervisory action. (11) Obtaining SARs. A State savings association may obtain SARs and the... SARs. SARs are confidential. Any institution or person subpoenaed or otherwise requested to disclose...

  14. 12 CFR 390.355 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Activity Report on the form prescribed by the FDIC. (3) SARs required. A State savings association shall... to supervisory action. (11) Obtaining SARs. A State savings association may obtain SARs and the... SARs. SARs are confidential. Any institution or person subpoenaed or otherwise requested to disclose...

  15. 12 CFR 563.180 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... under 12 CFR 510.5. (iv) Limitation on liability. A savings association or service corporation and any....C. 1813(u) and 1818(b)(9)). (iii) SAR means a Suspicious Activity Report. (3) SARs required. A... action. (11) Obtaining SARs. A savings association or service corporation may obtain SARs and...

  16. 12 CFR 563.180 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... under 12 CFR 510.5. (iv) Limitation on liability. A savings association or service corporation and any....C. 1813(u) and 1818(b)(9)). (iii) SAR means a Suspicious Activity Report. (3) SARs required. A... action. (11) Obtaining SARs. A savings association or service corporation may obtain SARs and...

  17. 12 CFR 563.180 - Suspicious Activity Reports and other reports and statements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... under 12 CFR 510.5. (iv) Limitation on liability. A savings association or service corporation and any....C. 1813(u) and 1818(b)(9)). (iii) SAR means a Suspicious Activity Report. (3) SARs required. A... action. (11) Obtaining SARs. A savings association or service corporation may obtain SARs and...

  18. A Fluorescent Reporter of AMPK activity and Cellular Energy Stress

    PubMed Central

    Tsou, Peiling; Zheng, Bin; Hsu, Chia-Hsien; Sasaki, Atsuo T; Cantley, Lewis C.

    2011-01-01

    SUMMARY AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here we describe a biosensor, AMPKAR, which exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatio-temporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 & α2 and on the upstream kinase, LKB1. Interestingly we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidics device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation. PMID:21459332

  19. Reportable Nuclide Criteria for ORNL Radioactive Waste Management Activities - 13005

    SciTech Connect

    McDowell, Kip; Forrester, Tim; Saunders, Mark

    2013-07-01

    The U.S. Department of Energy's Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee generates numerous radioactive waste streams. Many of those streams contain a large number of radionuclides with an extremely broad range of concentrations. To feasibly manage the radionuclide information, ORNL developed reportable nuclide criteria to distinguish between those nuclides in a waste stream that require waste tracking versus those nuclides of such minimal activity that do not require tracking. The criteria include tracking thresholds drawn from ORNL onsite management requirements, transportation requirements, and relevant treatment and disposal facility acceptance criteria. As a management practice, ORNL maintains waste tracking on a nuclide in a specific waste stream if it exceeds any of the reportable nuclide criteria. Nuclides in a specific waste stream that screen out as non-reportable under all these criteria may be dropped from ORNL waste tracking. The benefit of these criteria is to ensure that nuclides in a waste stream with activities which meaningfully affect safety and compliance are tracked, while documenting the basis for removing certain isotopes from further consideration. (authors)

  20. Reportable Nuclide Criteria for ORNL Waste Management Activities - 13005

    SciTech Connect

    McDowell, Kip; Forrester, Tim; Saunders, Mark Edward

    2013-01-01

    The U.S. Department of Energy's Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee generates numerous radioactive waste streams. Many of those streams contain a large number of radionuclides with an extremely broad range of concentrations. To feasibly manage the radionuclide information, ORNL developed a reportable nuclide criteria to distinguish between those nuclides in a waste stream that require waste tracking versus those nuclides of such minimal activity that do not require tracking. The criteria include tracking thresholds drawn from ORNL onsite management requirements, transportation requirements, and relevant treatment and disposal facility acceptance criteria. As a management practice, ORNL maintains waste tracking on a nuclide in a specific waste stream if it exceeds any of the reportable nuclide criteria. Nuclides in a specific waste stream that screen out as non-reportable under all these criteria may be dropped from ORNL waste tracking. The benefit of this criteria is to ensure that nuclides in a waste stream with activities which meaningfully affect safety and compliance are tracked, while documenting the basis for removing certain isotopes from further consideration.

  1. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  2. Activation of carbohydrate response element binding protein (ChREBP) by ethanol

    PubMed Central

    Liangpunsakul, Suthat; Ross, Ruth A.; Crabb, David W.

    2012-01-01

    Carbohydrate response element binding protein (ChREBP) is a transcription factor involved in hepatic lipogenesis. Its function is in part under the control of AMP-activated protein kinase (AMPK) and protein phosphatase 2A (PP2A). Given known effects of ethanol on AMPK and PP2A, it is plausible that ethanol might enhance fatty acid synthesis by increasing the activity of ChREBP. We hypothesized that another potential pathway of ethanol-induced hepatic steatosis is mediated by activation of ChREBP. Methods The effects of ethanol on ChREBP were assessed in hepatoma cells and in C57BL/6J mice fed with the Lieber-DeCarli diet. Results When the cells were exposed to ethanol (50 mM) for 24 hrs, the activity of a liver pyruvate kinase (LPK) promoter-luciferase reporter was increased by ~4-fold. Ethanol feeding of mice resulted in the translocation of ChREBP from cytosol to the nucleus. PP2A activity was increased in the liver of ethanol-fed mice by 22%. We found no difference in the levels of hepatic Xu-5-P between ethanol-fed mice and controls. Transfection of a constitutively active AMPK expression plasmid suppressed the basal activity of the LPK luciferase reporter and abolished the effect of ethanol on the reporter activity. However, transfection of rat hepatoma cells with a dominant negative AMPK expression plasmid induced basal LPK luciferase activity by only ~20%. The effect of ethanol on ChREBP was attenuated in the presence of okadaic acid, an inhibitor of PP2A. Conclusions The effects of ethanol on AMPK and PP2A may result in activation of ChREBP, providing another potential mechanism for ethanol-induced hepatic steatosis. However, additional okadaic acid-insensitive effects appear to be important as well. PMID:23266705

  3. Blocking the entrance of AMP pocket results in hormetic stimulation of imidazolium-based ionic liquids to firefly luciferase.

    PubMed

    Chen, Fu; Liu, Shu-Shen; Yu, Mo; Qu, Rui; Wang, Meng-Chao

    2015-08-01

    The hormesis characterized by low-concentration stimulation and high-concentration inhibition has gained significant interest over the past decades. Some organic solvents and ionic liquids (ILs) have hormetic concentration responses (HCR) to bioluminescence such as firefly luciferase and Vibrio qinghaiensis sp.-Q67. In this study, we determine the effects of 1-alkyl-3-methylimidazolium chlorine ILs ([Cnmim]Cl, n=2, 4, 6, 8, 10 and 12) to firefly luciferase in order to verify the mechanism of hormesis. The luminescence inhibition toxicity tests show that the stimulation effects of [C8mim]Cl and [C10mim]Cl are obvious, [C6mim]Cl and [C12mim]Cl are minor, and [C2mim]Cl and [C4mim]Cl are rare. The enzyme kinetics show that [C8mim]Cl and [C10mim]Cl are the competitive inhibitors with ATP while [C2mim]Cl and [C4mim]Cl are the noncompetitive ones. Molecular dynamics simulation results reveal that imidazolium rings of [C8mim] and [C10mim] locate at the entrance of luciferin pocket which is adjacent to AMP pocket, while alkyl-chains insert into the bottom of the luciferin pocket. Combining the results from inhibition test, kinetics assay and molecular simulation, we can deduce that occupying AMP pocket by imidazolium ring is responsible for hormetic stimulation. PMID:25835270

  4. Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Kobayashi, Naoki; Takakura, Yoshinobu

    2005-07-20

    Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells. PMID:15936841

  5. In Vivo Determination of Mitochondrial Function Using Luciferase-Expressing Caenorhabditis elegans: Contribution of Oxidative Phosphorylation, Glycolysis, and Fatty Acid Oxidation to Toxicant-Induced Dysfunction.

    PubMed

    Luz, Anthony L; Lagido, Cristina; Hirschey, Matthew D; Meyer, Joel N

    2016-01-01

    Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc. PMID:27479364

  6. Aryl hydrocarbon receptor-mediated activity of particulate organic matter from the Paso del Norte airshed along the U.S.-Mexico border.

    PubMed Central

    Arrieta, Daniel E; Ontiveros, Cynthia C; Li, Wen-Whai; Garcia, Jose H; Denison, Michael S; McDonald, Jacob D; Burchiel, Scott W; Washburn, Barbara Shayne

    2003-01-01

    In this study, we determined the biologic activity of dichloromethane-extracted particulate matter < 10 micro m in aerodynamic diameter (PM10) obtained from filters at three sites in the Paso del Norte airshed, which includes El Paso, Texas, USA; Juarez, Chihuahua, Mexico, and Sunland Park, New Mexico, USA. The extracts were rich in polycyclic aromatic hydrocarbons (PAHs) and had significant biologic activity, measured using two in vitro assay systems: ethoxyresorufin-(O-deethylase (EROD) induction and the aryl hydrocarbon-receptor luciferase reporter system. In most cases, both EROD (5.25 pmol/min/mg protein) and luciferase activities (994 relative light units/mg) were highest in extracts from the Advance site located in an industrial neighborhood in Juarez. These values represented 58% and 55%, respectively, of induction associated with 1 micro M ss-naphthoflavone exposures. In contrast, little activity was observed at the Northeast Clinic site in El Paso, the reference site. In most cases, luciferase and EROD activity from extracts collected from the Tillman Health Center site, situated in downtown El Paso, fell between those observed at the other two sites. Overall, a statistically significant correlation existed between PM10 and EROD and luciferase activities. Chemical analysis of extracts collected from the Advance site demonstrated that concentrations of most PAHs were higher than those reported in most other metropolitan areas in the United States. Calculations made with these data suggest a cancer risk of 5-12 cases per 100,000 people. This risk estimate, as well as comparisons with the work of other investigators, raises concern regarding the potential for adverse health effects to the residents of this airshed. Further work is needed to understand the sources, exposure, and effects of PM10 and particulate organic material in the Paso del Norte airshed. PMID:12896850

  7. Advanced Light Source activity report 1996/97

    SciTech Connect

    1997-09-01

    Ten years ago, the Advanced Light Source (ALS) existed as a set of drawings, calculations, and ideas. Four years ago, it stored an electron beam for the first time. Today, the ALS has moved from those ideas and beginnings to a robust, third-generation synchrotron user facility, with eighteen beam lines in use, many more in planning or construction phases, and hundreds of users from around the world. Progress from concepts to realities is continuous as the scientific program, already strong in many diverse areas, moves in new directions to meet the needs of researchers into the next century. ALS staff members who develop and maintain the infrastructure for this research are similarly unwilling to rest on their laurels. As a result, the quality of the photon beams the authors deliver, as well as the support they provide to users, continues to improve. The ALS Activity Report is designed to share the results of these efforts in an accessible form for a broad audience. The Scientific Program section, while not comprehensive, shares the breadth, variety, and interest of recent research at the ALS. (The Compendium of User Abstracts and Technical Reports provides a more comprehensive and more technical view.) The Facility Report highlights progress in operations, ongoing accelerator research and development, and beamline instrumentation efforts. Although these Activity Report sections are separate, in practice the achievements of staff and users at the ALS are inseparable. User-staff collaboration is essential as they strive to meet the needs of the user community and to continue the ALS's success as a premier research facility.

  8. Pterostilbene-mediated Nrf2 activation: Mechanistic insights on Keap1:Nrf2 interface.

    PubMed

    Bhakkiyalakshmi, Elango; Dineshkumar, Kesavan; Karthik, Suresh; Sireesh, Dornadula; Hopper, Waheeta; Paulmurugan, Ramasamy; Ramkumar, Kunka Mohanram

    2016-08-15

    The discovery of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors has become a promising strategy to develop novel lead molecules against variety of stress. Hence, Keap1-Nrf2 system plays an important role in oxidative/electrophilic stress associated disorders. Our earlier studies identified pterostilbene (PTS), a natural analogue of resveratrol, as a potent Nrf2 activator and Keap1-Nrf2 PPI inhibitor as assessed by luciferase complementation assay. In this study, we further identified the potential of PTS in Nrf2 activation and ARE-driven downstream target genes expression by nuclear translocation experiments and ARE-luciferase reporter assay, respectively. Further, the luciferase complementation assay identified that PTS inhibits Keap1-Nrf2 PPI in both dose and time-dependent manner. Computational studies using molecular docking and dynamic simulation revealed that PTS directly interacts with the basic amino acids of kelch domain of Keap1 and perturb Keap1-Nrf2 interaction pattern. This manuscript not only shows the binding determinants of Keap1-Nrf2 proteins but also provides mechanistic insights on Nrf2 activation potential of PTS. PMID:27312421

  9. Computer Science Research Institute 2004 annual report of activities.

    SciTech Connect

    DeLap, Barbara J.; Womble, David Eugene; Ceballos, Deanna Rose

    2006-03-01

    This report summarizes the activities of the Computer Science Research Institute (CSRI) at Sandia National Laboratories during the period January 1, 2004 to December 31, 2004. During this period the CSRI hosted 166 visitors representing 81 universities, companies and laboratories. Of these 65 were summer students or faculty. The CSRI partially sponsored 2 workshops and also organized and was the primary host for 4 workshops. These 4 CSRI sponsored workshops had 140 participants--74 from universities, companies and laboratories, and 66 from Sandia. Finally, the CSRI sponsored 14 long-term collaborative research projects and 5 Sabbaticals.

  10. Computer Science Research Institute 2005 annual report of activities.

    SciTech Connect

    Watts, Bernadette M.; Collis, Samuel Scott; Ceballos, Deanna Rose; Womble, David Eugene

    2008-04-01

    This report summarizes the activities of the Computer Science Research Institute (CSRI) at Sandia National Laboratories during the period January 1, 2005 to December 31, 2005. During this period, the CSRI hosted 182 visitors representing 83 universities, companies and laboratories. Of these, 60 were summer students or faculty. The CSRI partially sponsored 2 workshops and also organized and was the primary host for 3 workshops. These 3 CSRI sponsored workshops had 105 participants, 78 from universities, companies and laboratories, and 27 from Sandia. Finally, the CSRI sponsored 12 long-term collaborative research projects and 3 Sabbaticals.

  11. FY15 Status Report on NEAMS Neutronics Activities

    SciTech Connect

    Lee, C. H.; Shemon, E. R.; Smith, M. A.; Connaway, H. M.; Aliberti, G.

    2015-09-30

    This report summarizes the current status of NEAMS activities in FY2015. The tasks this year are (1) to improve solution methods for steady-state and transient conditions, (2) to develop features and user friendliness to increase the usability and applicability of the code, (3) to improve and verify the multigroup cross section generation scheme, (4) to perform verification and validation tests of the code using SFRs and thermal reactor cores, and (5) to support early users of PROTEUS and update the user manuals.

  12. Activity Report, 1990 - 1992 and proceedings, volume 2

    NASA Astrophysics Data System (ADS)

    Kottnauer, Ing. Pavel

    A report of the European Seismological Commission (ESC) on 1990-1992 activities and Proceedings of the General Assembly of the ESC are presented in two volumes. Volume 2 covers the following topics: study of seismic sound, seismotectonic analysis, deep seismic sounding, the three-dimensional structure of the European lithosphere-asthenosphere system, complexity in earthquake occurrence, earthquake hazard, strong and weak earthquake ground motions, macroseismology, microzonation, and applications in earthquake engineering. One paper dealing with the connection between seismicity and the CO2- (sup 222)Rn content in spring water has been inputted to INIS.

  13. Activity report 1990-1992 and proceedings, volume 1

    NASA Astrophysics Data System (ADS)

    Mayer-Rosa, D.; Waniek, L.; Suhadolc, P.

    A report on the activities of the European Seismological Commission (ESC) during the period 1990-1992 together with the Proceedings of the General Assembly of the ESC are presented in two volumes. Volume 1 covers the following topics: recent strong earthquakes in Europe, regional seismicity, historical earthquakes in Europe, statistical models and methods in seismology, numerical modeling in three-dimensional media, methodology of quantification of European earthquakes and recent results, seismic noise and signal detectability, regional seismic network, and history of seismometry. One paper dealing with microseismic noise characteristics around the Kozloduy nuclear power plant has been inputted to INIS.

  14. Computer Science Research Institute 2003 annual report of activities.

    SciTech Connect

    DeLap, Barbara J.; Womble, David Eugene; Ceballos, Deanna Rose

    2006-03-01

    This report summarizes the activities of the Computer Science Research Institute (CSRI) at Sandia National Laboratories during the period January 1, 2003 to December 31, 2003. During this period the CSRI hosted 164 visitors representing 78 universities, companies and laboratories. Of these 78 were summer students or faculty members. The CSRI partially sponsored 5 workshops and also organized and was the primary host for 3 workshops. These 3 CSRI sponsored workshops had 178 participants--137 from universities, companies and laboratories, and 41 from Sandia. Finally, the CSRI sponsored 18 long-term collaborative research projects and 5 Sabbaticals.

  15. Frio II Brine Pilot: Report on GEOSEQ Activities

    SciTech Connect

    Daley, T.M.; Freifeld, B.M.; Ajo-Franklin, J.B.; Doughty, C.; Benson, S.M.

    2007-11-17

    LBNL's GEOSEQ project is a key participant in the Frio IIbrine pilot studying geologic sequestration of CO2. During During theinjection phase of the Frio-II brine pilot, LBNL collected multiple datasets including seismic monitoring, hydrologic monitoring and geochemicalsampling. These data sets are summarized in this report including allCASSM (continuous active source seismic monitoring) travel time data,injection pressure and flow rate data and gaseous sampling and tracerdata. Additional results from aqueous chemistry analysis performed by theU. S. Geological Survey (USGS) are summarized. Post injectionmodification of the flow model for Frio II is shown. Thesemodificationsare intended to facilitate integration with the monitoring data andincorporation of model heterogeneity. Current activities of LBNL's GEOSEQproject related to the Frio II test are shown, including development of anew petrophysical model for improved interpretation of seismic monitoringdata and integration of this data with flow modeling.

  16. CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1.

    PubMed

    Yang, Pei-Ming; Lin, Pei-Jie; Chen, Ching-Chow

    2012-04-01

    CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1. PMID:22419072

  17. Stanford Synchrotron Radiation Laboratory activity report for 1987

    SciTech Connect

    Robinson, S.; Cantwell, K.

    1988-12-31

    During 1987, SSRL achieved many significant advances and reached several major milestones utilizing both SPEAR and PEP as synchrotron radiation sources as described in this report. Perhaps the following two are worthy of particular mention: (1) SPEAR reached an all time high of 4,190 delivered user-shifts during calendar year 1987, highlights of the many scientific results are given; (2) during a 12 day run in December of 1987, PEP was operated in a low emittance mode (calculated emittance 6.4 nanometer-radians) at 7.1 GeV with currents up to 33 mA. A second undulator beam line on PEP was commissioned during this run and used to record many spectra showing the extremely high brightness of the radiation. PEP is now by far the highest brightness synchrotron radiation source in the world. The report is divided into the following sections: (1) laboratory operations; (2) accelerator physics programs; (3) experimental facilities; (4) engineering division; (5) conferences and workshops; (6) SSRL organization; (7) experimental progress reports; (8) active proposals; (9) SSRL experiments and proposals by institution; and (10) SSRL publications.

  18. Cytocidal activities of topoisomerase 1 inhibitors and 5-azacytidine against pheochromocytoma/paraganglioma cells in primary human tumor cultures and mouse cell lines.

    PubMed

    Powers, James F; Korgaonkar, Parimal G; Fliedner, Stephanie; Giubellino, Alessio; Pacak, Karel; Sahagian, G Gary; Tischler, Arthur S

    2014-01-01

    There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and timing of their

  19. Cytocidal Activities of Topoisomerase 1 Inhibitors and 5-Azacytidine against Pheochromocytoma/Paraganglioma Cells in Primary Human Tumor Cultures and Mouse Cell Lines

    PubMed Central

    Powers, James F.; Korgaonkar, Parimal G.; Fliedner, Stephanie; Giubellino, Alessio; Sahagian, Karel Pacak, G. Gary.; Tischler, Arthur S.

    2014-01-01

    There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in current chemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells have therefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere with mechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary cultures of representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to show that efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministration of sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested the same drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivo drug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased by both camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromogranin A, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferase and GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof of principle for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancing efficacy and reducing toxicity by optimizing the combination and timing of their

  20. Capturing community change: Active Living by Design's progress reporting system.

    PubMed

    Bors, Philip A

    2012-11-01

    The Active Living by Design (ALbD) National Program Office (NPO) developed an evaluation system to track progress of 25 community partnerships, funded by the Robert Wood Johnson Foundation (RWJF). Between June 2004 and October 2008, partnerships documented their actions and accomplishments through ALbD's online Progress Reporting System (PRS) database. All entries were verified and analyzed by the NPO. Results from the PRS suggest that the ALbD partnerships were successful fundraisers, leveraging $256 million from grants, policy decisions, in-kind and direct sources. The partnerships also documented newspaper coverage, TV, and radio air time and they developed physical activity programs such as exercise clubs and "walking school buses." Partnerships were adept at influencing decision makers to create or rewrite policies and improve built environments. Selected policy examples included, but were not limited to, approvals for capital improvements, street design standards, and development ordinances. Partnerships also contributed to the completion and approval of influential planning products, such as comprehensive land use, neighborhood, and roadway corridor plans. The most common built-environment changes were street improvements for safer pedestrian and bicycle travel, including new crosswalks, bicycle facilities, and sidewalks. The ALbD community partnerships' accomplishments and challenges contribute to knowledge and best practices in the active living field. Five years after their grant began, RWJF's initial investment showed substantial and measurable results. PMID:23079260

  1. Report of activities performed, July 1, 1980-June 30, 1981

    SciTech Connect

    Not Available

    1981-01-01

    The Numerical Data Advisory Board (NDAB) is a body within the National Research Council composed of a general board with specialized committees and panels. The objective of NDAB and its committees and panels is the improvement in quality, reliability, availability, accessibility, dissemination, utilization, and management of data. NDAB seeks to promote an appreciation of the importance of evaluated data to scientists, engineers, regulators, and others who require reliable numerical data for research and for decision making. NDAB is an interdisciplinary body with representation from physical, chemical, engineering, biological, and geological sciences. Selected sociotechnical, socioeconomic, and transient, or soft data topics are also covered. An effective path of communication with international data activities is maintained by scheduling NDAB meetings jointly with the US National Committee for CODATA, the Committee on Data for Science and Technology of the International Council for Scientific Unions (ICSU). An active government liaison relationship is maintained to facilitate input from, and discussion with branches of agencies that deal with technical data and information programs. NDAB has addressed both broad, generic cross-cutting data problems pertinent to all agencies that support R and D programs, as well as specific issues. For some of the specific topics, ad hoc meetings with subgroups of NDAB and the specific agencies requesting such discussions were held. Meetings held by NDAB for the time period covered by this report, as well as other activities, are summarized in Attachment A.

  2. [On the Effect of α-Tocopherol on Protein Kinase C Activity in vitro].

    PubMed

    Krassova, N E; Ugraitskaya, S V; Penkov, N V; Fesenko, E E

    2015-01-01

    The effect of the antioxidant α-tocopherol on rat brain protein kinase C activity as a model to study bimodal dose-dependent effect has been investigated. Enzyme activity has been monitored photometrically with a luciferase reporter assay that measures ADP produced by posphorylation. The inhibition of protein kinase C activity by α-tocopherol was found at the concentration range from 10(-3) to 10(-6) M with no effect of ultra low doses of the antioxidant (below. 10(-12) M). The absence of bimodal dose-dependent effect may be associated with the enzyme source. PMID:26591616

  3. A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

  4. 2001 NSLS ACTIVITY REPORT (NATIONAL SYNCHROTRON LIGHT SOURCE).

    SciTech Connect

    CORWIN, M.A.

    2002-05-01

    The year 2001 has been another highly productive year at the NSLS, with over 2500 users, including 720 first time users, conducting nearly 1200 experiments in fields ranging from the life, materials, chemical, and environmental sciences to applied science and technology. An impressive array of highlights from this scientific activity is included in this Activity Report. They include the first demonstration of a direct structural probe of the superconducting ground state in the cuprates by utilizing anomalous soft x-ray resonance effects to selectively enhance the scattering from doped holes. Another highly significant result was the determination of the structure of the potassium channel membrane protein. This is especially significant as it provides insight into how the channel functions and how it selects a particular kind of ion. In the nanoscience area, small angle x-ray scattering measurements played an essential role in determining that preferential sequestering of tailored metal nanocrystals into a self-assembled lamellar diblock copolymer can produce high quality metallodielectric photonic bandgap structures, demonstrating the potential of these nanocomposites for photonic crystal engineering. The infrared microscopy program continued to yield noteworthy results, including an important study that characterized the types and abundances of organic materials in contaminated and uncontaminated sediments from the New York/New Jersey Harbor. These results will be useful in devising improved methods for the destruction or removal of these environmental contaminants.

  5. Status report on the survey and alignment activities at Fermilab

    SciTech Connect

    Oshinowo, Babatunde O'Sheg; /Fermilab

    2004-10-01

    The surveying and alignment activities at Fermilab are the responsibility of the Alignment and Metrology Group. The Group supports and interacts with physicists and engineers working on any particular project, from the facility construction phase to the installation and final alignment of components in the beam line. One of the goals of the Alignment and Metrology Group is to upgrade the old survey networks in the tunnel using modern surveying technology, such as the Laser Tracker for tunnel networks and GPS for the surface networks. According to the job needs, all surveys are done with Laser Trackers and/or Videogrammetry (V-STARS) systems for spatial coordinates; optical and electronic levels are used for elevations, Gyro-Theodolite for azimuths, Mekometer for distances and GPS for baseline vectors. The group has recently purchased two new API Laser Trackers, one INCA3 camera for the V-Stars, and one DNA03 digital level. This report presents the projects and major activities of the Alignment and Metrology Group at Fermilab during the period of 2000 to 2004. It focuses on the most important current projects, especially those that have to be completed during the currently scheduled three-month shutdown period. Future projects, in addition to the status of the current projects, are also presented.

  6. Report on active and planned spacecraft and experiments

    NASA Technical Reports Server (NTRS)

    Schofield, N. J., Jr.; Littlefield, R. G.; Elsen, M. F.

    1985-01-01

    This report provides the professional community with information on current and planned spacecraft activity (including both free-flying spacecraft and Shuttle-attached payloads) for a broad range of scientific disciplines. By providing a brief description of each spacecraft and experiment as well as its current status, it is hoped that this document will be useful to many people interested in the scientific, applied, and operational uses of the data collected. Furthermore, for those investigators who are planning or coordinating future observational programs employing a number of different techniques such as rockets, balloons, aircraft, ships, and buoys, this document can provide some insight into the contributions that may be provided by orbiting instruments. The document includes information concerning active and planned spacecraft and experiments. The information covers a wide range of scientific disciplines: astronomy, earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. These spacecraft projects represent the efforts and funding of individual countries, as well as cooperative arrangements among different countries.

  7. Forecast for solar cycle 23 activity: a progress report

    NASA Astrophysics Data System (ADS)

    Ahluwalia, H. S.

    2001-08-01

    At the 25th International Cosmic Ray Conference (ICRC) at Durban, South Africa, I announced the discovery of a three cycle quasi-periodicity in the ion chamber data string assembled by me, for the 1937 to 1994 period (Conf. Pap., v. 2, p. 109, 1997). It corresponded in time with a similar quasi-periodicity observed in the dataset for the planetary index Ap. At the 26th ICRC at Salt Lake City, UT, I reported on our analysis of the Ap data to forecast the amplitude of solar cycle 23 activity (Conf. Pap., v. 2, pl. 260, 1999). I predicted that cycle 23 will be moderate (a la cycle 17), notwithstanding the early exuberant forecasts of some solar astronomers that cycle 23, "may be one of the greatest cycles in recent times, if not the greatest." Sunspot number data up to April 2001 indicate that our forecast appears to be right on the mark. We review the solar, interplanetary and geophysical data and describe the important lessons learned from this experience. 1. Introduction Ohl (1971) was the first to realize that Sun may be sending us a subliminal message as to its intent for its activity (Sunspot Numbers, SSN) in the next cycle. He posited that the message was embedded in the geomagnetic activity (given by sum Kp). Schatten at al (1978) suggested that Ohl hypothesis could be understood on the basis of the model proposed by Babcock (1961) who suggested that the high latitude solar poloidal fields, near a minimum, emerge as the toroidal fields on opposite sides of the solar equator. This is known as the Solar Dynamo Model. One can speculate that the precursor poloidal solar field is entrained in the high speed solar wind streams (HSSWS) from the coronal holes which are observed at Earth's orbit during the descending phase of the previous cycle. The interaction

  8. 77 FR 75198 - Standard Format and Content for Post-Shutdown Decommissioning Activities Report

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-19

    ... COMMISSION Standard Format and Content for Post-Shutdown Decommissioning Activities Report AGENCY: Nuclear... Format and Content for Post-shutdown Decommissioning Activities Report.'' This guide describes a method...) 1.185, ``Standard Format and Content for Post-shutdown Decommissioning Activities Report,''...

  9. Annual Report on Activities, Israel Science Teaching Centre 1971-1972.

    ERIC Educational Resources Information Center

    Amos de Shalit Science Teaching Centre (Israel).

    This is the annual report of the activities of Israel Science Teaching Centre for 1971-1972. The contents of the report include reports of meetings and activities undertaken in various projects in mathematics, physics, chemistry, biology, agriculture, and the junior school program. Some major activities of different projects include inservice…

  10. Activities report of the Centro Studi E Laboratori Telecomunicazioni: Reports and balances

    NASA Astrophysics Data System (ADS)

    1991-05-01

    The CSELT management report on structure and activities covers public and ISDN (Integrated Services Digital Network) networks, intelligence and service networks, mobile services, management of OSS (Operation Support Systems) and networks, high-speed networks and services, optical networks and systems, radio points and satellites, and duality and conformity tests. Management progress, investments, personnel, participation in European projects, and collaboration with Italian universities are outlined. An analysis of income results and of the patrimonial and financial situations is given. Balances at 31 Dec. 1990 are shown.

  11. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  12. Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

    PubMed

    Ramirez, Tzutzuy; Stein, Nadine; Aumann, Alexandra; Remus, Tina; Edwards, Amber; Norman, Kimberly G; Ryan, Cindy; Bader, Jackie E; Fehr, Markus; Burleson, Florence; Foertsch, Leslie; Wang, Xiaohong; Gerberick, Frank; Beilstein, Paul; Hoffmann, Sebastian; Mehling, Annette; van Ravenzwaay, Bennard; Landsiedel, Robert

    2016-04-01

    Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers. PMID:26796489

  13. Special Report. Federal Activities Addressing Violence in Schools.

    ERIC Educational Resources Information Center

    Barrios, Lisa C., Comp.; Baer, Katie; Bennett, Gwendolyn; Bergan, Anne; Bryn, Stephanie; Callaway, Sue; Davis, Darlind; Downs, Ray; Dressler, Kellie; Ho, Tiffany; Karp, Naomi; Mathews-Younes, Anne; MacMurray, Nataki; O'Brien, Eileen; Overpeck, Mary; Reed, Winnie; Small, Meg; Tuma, Farris

    2000-01-01

    Presents an inventory of federal activities that address violence in schools. Agencies were asked to identify all ongoing activities and recently completed efforts regarding school violence. The resulting categories include: surveillance activities, evaluation research activities, other research activities, research synthesis and application…

  14. Stanford Synchrotron Radiation Laboratory. Activity report for 1988

    SciTech Connect

    Cantwell, K.

    1996-01-01

    For SSRL operations, 1988 was a year of stark contrasts. The first extended PEP parasitic running since the construction of our two beam lines on that storage ring took place in November and December. Four experiments discussed below, were performed and detailed operational procedures which allowed synchrotron radiation an high energy users to coexist were established. SSRL anticipates that there will be significant amounts of beam time when PEP is run again for high energy physics. On the other hand, activity on SPEAR consisted of brief parasitic running on the VUV lines in December when the ring was operated at 1.85 GeV for colliding beam experiments. There was no dedicated SPEAR running throughout the entire calendar year. This is the first time since dedicated SPEAR operation was initiated in 1980 that there was no such running. The decision was motivated by both cost and performance factors, as discussed in Section 1 of this report. Fortunately, SLAC and SSRL have reached an agreement on SPEAR and PEP dedicated time charges which eliminates the cost volatility which was so important in the cancellation of the June-July dedicated SPEAR run. As discussed in Section 2, the 3 GeV SPEAR injector construction is proceeding on budget and on schedule. The injector will overcome the difficulties associated with the SLC-era constraint of only two injections per day. SSR and SLAC have also embarked on a program to upgrade SPEAR to achieve high reliability and performance. As a consequence, SSRL`s users may anticipate a highly effective SPEAR by 1991, at the latest. At that time, SPEAR is expected to be fully dedicated to synchrotron radiation research and operated by SSRL. Also contained in this report is a discussion of the improvements to SSRL`s experimental facilities and highlights of the experiments of the past year.

  15. Aeronautics and space report of the President: 1981 activities

    NASA Technical Reports Server (NTRS)

    1981-01-01

    Achievements in the aeronautics and space program by function are summarized. Activities in communications, Earth's resources and environment, space science, space transportation, international activities, and aeronautics are included.

  16. RELAP-7 Progress Report. FY-2015 Optimization Activities Summary

    SciTech Connect

    Berry, Ray Alden; Zou, Ling; Andrs, David

    2015-09-01

    This report summarily documents the optimization activities on RELAP-7 for FY-2015. It includes the migration from the analytical stiffened gas equation of state for both the vapor and liquid phases to accurate and efficient property evaluations for both equilibrium and metastable (nonequilibrium) states using the Spline-Based Table Look-up (SBTL) method with the IAPWS-95 properties for steam and water. It also includes the initiation of realistic closure models based, where appropriate, on the U.S. Nuclear Regulatory Commission’s TRACE code. It also describes an improved entropy viscosity numerical stabilization method for the nonequilibrium two-phase flow model of RELAP-7. For ease of presentation to the reader, the nonequilibrium two-phase flow model used in RELAP-7 is briefly presented, though for detailed explanation the reader is referred to RELAP-7 Theory Manual [R.A. Berry, J.W. Peterson, H. Zhang, R.C. Martineau, H. Zhao, L. Zou, D. Andrs, “RELAP-7 Theory Manual,” Idaho National Laboratory INL/EXT-14-31366(rev. 1), February 2014].

  17. Report on research activities for calendar year 1990

    SciTech Connect

    Patrick, W.C.; Ababou, R.; Chowdhury, A.H.; Cragnolino, G.; Dodge, F.T.; Green, R.T.; Gureghian, A.B.; Hsiung, S.M.; Murphy, W.M.; Pabalan, R.T.; Pearch, E.C.; Sagar, B.; Sridhar, N. . Center for Nuclear Waste Regulatory Analyses)

    1991-12-01

    This is an annual status report the results of research conducted on behalf of the NRC by the Center for Nuclear Waste Regulatory Analyses in support of activities under the Nuclear Waste Policy Act, as Amended. Eight specific projects are underway. The Geochemistry project is using laboratory methods and computer calculations to assess key geochemical constrains and to evaluate sorptive properties of zeolite present at the proposed repository site. The Thermohydrology project has as its focus improved understanding of heat and fluid flow in unsaturated media. Laboratory, field, and calculational studies are combined in the Seismic Rock Mechanics project to examine the effects of repeated seismic loadings on the rock-mechanical and hydrological responses of rock masses. The Integrated Waste Package Experiments have been initiated to evaluate degradation modes of candidate waste container alloys. Three-dimensional computer analysis techniques are being used to investigate spatial variability of flow and transport in variably saturated fractured porous media in the Stochastic Flow and Transport project. The recently initiated Geochemical Analogs project seeks to investigate the role of such analogs in the licensing process, and is currently focused on locating and evaluating a potential site for investigation. The Performance Assessment project is directed toward developing and evaluating methodologies for evaluation of the long-term performance of the proposed repository.

  18. GENERATION OF TWO STABLE CELL LINES THAT EXPRESS HER-ALPHA OR HER-ALPHA AND -BETA AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

    EPA Science Inventory

    Generation of Two Stable Cell Lines that Express hERa or
    hERa and b and Firefly Luciferase Genes for Endocrine Screening

    K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1

    1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reprod...

  19. In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

    PubMed Central

    Sommer, J M; Cheng, Q L; Keller, G A; Wang, C C

    1992-01-01

    The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes. Images PMID:1515676

  20. Application of the luciferin-luciferase enzyme system for determination of adenosine triphosphate (ATP) to studies on the mechanisms of herbicide action

    NASA Technical Reports Server (NTRS)

    St.john, J. B.

    1975-01-01

    The luciferin-luciferase enzyme system for determination of ATP is valuable for studies on the mechanisms of herbicide action. Investigations using this system have shown that certain herbicides may act by interfering with ATP production or by blocking ATP use, or by both mechanisms.

  1. A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

    PubMed

    Amaral, Danilo T; Oliveira, Gabriela; Silva, Jaqueline R; Viviani, Vadim R

    Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation. PMID:27454752

  2. Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.

    PubMed

    Sonamoto, Rie; Kii, Isao; Koike, Yuka; Sumida, Yuto; Kato-Sumida, Tomoe; Okuno, Yukiko; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2015-01-01

    The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases. PMID:26234946

  3. A Measurable Activation of the bZIP Transcription Factor Atf1 in a Fission Yeast Strain Devoid of Stress-activated and Cell Integrity Mitogen-activated Protein Kinase (MAPK) Activities*

    PubMed Central

    Zhou, Xin; Ma, Yan; Kato, Toshiaki; Kuno, Takayoshi

    2012-01-01

    In Schizosaccharomyces pombe, the stress-activated Sty1 MAPK pathway is essential for cell survival under stress conditions. The Sty1 MAPK regulates Atf1 transcription factor to elicit stress responses in extreme conditions of osmolarity and reactive oxygen species-generating agents such as hydrogen peroxide, heat, low glucose, and heavy metal. Herein, using a newly developed Renilla luciferase reporter assay with enhanced detection sensitivity and accuracy, we show that distinct signaling pathways respond to cadmium and other reactive oxygen species-generating agents for the activation of Atf1. Also, surprisingly, a measurable activation of Atf1 transcription factor was still observed devoid of Sty1 MAPK activity. Further genetic and biological analyses revealed that the residual activation is caused by the activation of the cell wall integrity Pmk1 MAPK pathway and a redox-mediated activation of Atf1. PMID:22661707

  4. Development of a luciferase based viral inhibition assay to evaluate vaccine induced CD8 T-cell responses

    PubMed Central

    Naarding, Marloes A.; Fernandez-Fernandez, Natalia; Kappes, John C.; Hayes, Peter; Ahmed, Tina; Icyuz, Mert; Edmonds, Tara G.; Bergin, Philip; Anzala, Omu; Hanke, Tomas; Clark, Lorna; Cox, Josephine H.; Cormier, Emmanuel; Ochsenbauer, Christina; Gilmour, Jill

    2014-01-01

    Emergence of SIV and HIV specific CD8 T cells has been shown to correlate with control of in vivo replication. Poor correlation between IFN-γ ELISPOT responses and in vivo control of the virus has triggered the development of more relevant assays to assess functional HIV-1 specific CD8 T-cell responses for the evaluation and prioritization of new HIV-1 vaccine candidates. We previously established a viral inhibition assay (VIA) that measures the ability of vaccine-induced CD8 T-cell responses to inhibit viral replication in autologous CD4 T cells. In this assay, viral replication is determined by measuring p24 in the culture supernatant. Here we describe the development of a novel VIA, referred to as IMC LucR VIA that exploits replication-competent HIV-1 infectious molecular clones (IMCs) in which the complete proviral genome is strain-specific and which express the Renilla luciferase (LucR) gene to determine viral growth and inhibition. The introduction of the luciferase readout does provide significant improvement of the read out time. In addition to switching to the LucR read out, changes made to the overall protocol resulted in the miniaturization of the assay from a 48 to a 96-well plate format, which preserved sample and allowed for the introduction of replicates. The overall assay time was reduced from 13 to 8 days. The assay has a high degree of specificity, and the previously observed non-specific background inhibition in cells from HIV-1 negative volunteers has been reduced dramatically. Importantly, we observed an increase in positive responses, indicating an improvement in sensitivity compared to the original VIA. Currently, only a limited number of “whole-genome” IMC-LucR viruses are available and our efforts will focus on expanding the panel to better evaluate anti-viral breadth. Overall, we believe the IMC LucR VIA provides a platform to assess functional CD8 T-cell responses in large-scale clinical trial testing, which will enhance the ability

  5. Characteristics of a thyroid hormone responsive reporter gene transduced into a Xenopus laevis cell line using lentivirus vector.

    PubMed

    Sugiyama, Shin-Ichiro; Miyoshi, Hiroyuki; Yamauchi, Kiyoshi

    2005-12-01

    We introduced a self-inactivation (SIN) lentivirus vector (LV) into Xenopus laevis cell lines and established a permanent cell line expressing a reporter gene in a 3,5,3'-l-triiodothyronine (T(3)) dependent manner. The SIN LV contained the luciferase gene downstream from the X. laevis T(3)-response elements (TREs) and the SV40 promoter, and the enhanced green fluorescent protein (EGFP) gene downstream from the cytomegalovirus (CMV) promoter. It was integrated into the genome of X. laevis XL58, XTC2, and KR cells. The SIN LV transduced the X. laevis cells as efficiently as mammalian cells; however, the expression of EGFP in the transgene decreased with increasing culture time. A cell clone exhibiting the highest TH-dependent luciferase gene expression (XL58-TRE-Luc clone) was isolated from the EGFP-positive XL58 cell pool and characterized. The minimum effective concentration of T(3) that significantly induced the luciferase gene expression was 10(-11)M in the XL58-TRE-Luc clone. The application of the luciferase gene assay using the permanent XL58-TRE-Luc clone for the screening of thyroid-disrupting chemicals revealed that tetrachlorobisphenol A, at 10(-6)M, had a weak T(3)-agonist activity, whereas trichlorobisphenol A, at 10(-8) - 10(-6)M had a weak T(3)-antagonist activity. Our results indicated that the permanent X. laevis cell line containing a T(3)-response transgene could be used as a bioassay, with small intra-assay variation, for the rapid screening, identification, and characterization of the thyroid-disrupting chemicals. PMID:16102758

  6. 2015 Summary Report on Industrial and Regulatory Engagement Activities

    SciTech Connect

    Thomas, Kenneth David

    2015-09-01

    The Advanced Instrumentation, Information, and Control (II&C) Systems Technologies pathway of the Light Water Reactor Sustainability(LWRS) Program conducts a vigorous engagement strategy with the U.S. nuclear power industry, including the nuclear operating companies, major support organizations, the Nuclear Regulatory Commission (NRC), and suppliers. The goal of this engagement strategy is to develop a shared vision and common understanding across the nuclear industry of the need for II&C modernization, the performance improvement that can be obtained, and the opportunities for collaboration to enact this vision. The primary means of engaging the nuclear operating companies is through a Utility Working Group (UWG), composed of utility representatives that participate in formal meetings and bi-monthly phone calls to provide input on nuclear plant needs and priorities for II&C technologies. Two working groups were initiated during FY 2015 to provide a means for UWG members to focus on particular technologies of interest. The Outage Improvement Working Group consists of eight utilities that participate in periodic conference calls and have access to a share-point web page for acccess to project materials developed in the Advanced Outage Control Center pilot project. In the area of computer-based procedures and automated work packages, the II&C Pathway has worked with the Nuclear Information Technology Strategic Leadership (NITSL) to set up a monthly conference call with interested utility members to discuss various aspects of mobile worker technologies. Twenty one technical and project reports were delivered to the UWG during FY 2015, reflecting the work of the II&C Pathway pilot projects during the year. Distribution of these reports is one of the primary means of transferring to the nuclear industry the knowledge and experience gained during the development of advanced II&C technologies in support of LWR sustainability. Site visits to discuss pilot project

  7. Report on the Activities of National Balloon Facility, Hyderabad

    NASA Astrophysics Data System (ADS)

    Vasudevan, Rajagopalan; Sreenivasan, S.; Suneel Kumar, B.; Kulkarni, P. M.

    2012-07-01

    More than five and half decades back, the Indian Balloon Group at Tata Institute of Fundamental Research, Mumbai started development of stratospheric zero pressure balloon technology and today it is one among the leading balloon groups in the world. For the past 40 years, the Institute has been operating a Scientific Balloon Facility at Hyderabad and carried out 478 balloon flights for various disciplines of space sciences like primary cosmic ray studies, X ray, Gamma Ray, Infra Red Astronomies and Atmospheric science maintaining 100% success rate during the past nine years. The Balloon Facility has the capability to build balloons of volume up to 750,000 Cu.M. as well as carrying out R & D in all aspects of scientific ballooning like balloon engineering, balloon material development, general and flight support instrumentation. A continued effort in R & D for ultra thin balloon material for High Altitude Sounding Flights has resulted in lowering the thickness of the proven indigenous Antrix film initially from 6 to 3.8 microns in the first phase and further reduction to 2.7 microns in the second phase. A test balloon of volume 5000 Cu.M. using the 2.7 micron film attained a record altitude of 45.0 Km. amsl with 1 Kg. GPS sonde payload. A 60,000 Cu.M. balloon fabricated out of 3.8 micron film capable of reaching 47 Km. Altitude with 10 Kg. Payload is awaiting trial. This report briefly describes our balloon activities during the past two years. In atmospheric sciences, aerosol studies were made with OPC,QCM,Aethelometer, Nephelometer,MWR, CIMEL Sun Photometer and Raman LIDAR.Measuments of vertical profile of Meteorological parameters and ozone upto stratosphere using GPS Radiosonde and Ozone sonde is made respectively.Study of Ionospheric tomography is done with CADI and CRABEX.

  8. Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

    PubMed Central

    Liu, Xin; Noha, Stefan M.; Malainer, Clemens; Kramer, Matthias P.; Cocic, Amina; Kunert, Olaf; Schinkovitz, Andreas; Heiss, Elke H.; Schuster, Daniela

    2013-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements. PMID:23630612

  9. 78 FR 3029 - Comment Request for Information Collection for the Trade Activity Participant Report (TAPR...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-15

    ... Participant Report (TAPR); Extension Without Revisions AGENCY: Employment and Training Administration (ETA... collection of data about The Trade Activity Participant Report (OMB No. 1205-0392), which provides... changes. Title: Trade Activity Participant Report. OMB Number: 1205-0392. Affected Public: State, Local...

  10. 40 CFR 710.50 - Activities for which reporting is not required.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Activities for which reporting is not... SUBSTANCES CONTROL ACT TSCA CHEMICAL INVENTORY REGULATIONS Inventory Update Reporting for 2006 and Beyond § 710.50 Activities for which reporting is not required. A person described in § 710.48 is not...

  11. 40 CFR 710.30 - Activities for which reporting is not required.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Activities for which reporting is not... SUBSTANCES CONTROL ACT TSCA CHEMICAL INVENTORY REGULATIONS 2002 Inventory Update Reporting § 710.30 Activities for which reporting is not required. A person described in § 710.28 is not subject to...

  12. An MENC Bicentennial Commission Report on State Activities

    ERIC Educational Resources Information Center

    Music Educators Journal, 1976

    1976-01-01

    The MENC Bicentennial Commission requested each state chairman to submit a list of projects that were representative of the activities in that state. Included here are 27 states and the music activities appropriate for the Bicentennial year. (Editor/RK)

  13. Mass spectrometry analysis and transcriptome sequencing reveal glowing squid crystal proteins are in the same superfamily as firefly luciferase.

    PubMed

    Gimenez, Gregory; Metcalf, Peter; Paterson, Neil G; Sharpe, Miriam L

    2016-01-01

    The Japanese firefly squid Hotaru-ika (Watasenia scintillans) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1-3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1-3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates. PMID:27279452

  14. Mass spectrometry analysis and transcriptome sequencing reveal glowing squid crystal proteins are in the same superfamily as firefly luciferase

    PubMed Central

    Gimenez, Gregory; Metcalf, Peter; Paterson, Neil G.; Sharpe, Miriam L.

    2016-01-01

    The Japanese firefly squid Hotaru-ika (Watasenia scintillans) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1–3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1–3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates. PMID:27279452

  15. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase immunoprecipitation system.

    PubMed

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR. PMID:26506137

  16. Recommendations to improve the accuracy of estimates of physical activity derived from self report

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Assessment of physical activity using self-report has the potential for measurement error that can lead to incorrect inferences about physical activity behaviors and bias study results. To provide recommendations to improve the accuracy of physical activity derived from self report. We provide an ov...

  17. Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development.

    PubMed

    Choi, Yoori; Hwang, Do Won; Kim, Mee Young; Kim, Joo Yeon; Sun, Woong; Lee, Dong Soo

    2016-01-01

    MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs. PMID:27462205

  18. Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development

    PubMed Central

    Choi, Yoori; Hwang, Do won; Kim, Mee Young; Kim, Joo Yeon; Sun, Woong; Lee, Dong Soo

    2016-01-01

    MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs. PMID:27462205

  19. Active Matrix Organic Light Emitting Diode (AMOLED) Environmental Test Report

    NASA Technical Reports Server (NTRS)

    Salazar, George A.

    2013-01-01

    This report focuses on the limited environmental testing of the AMOLED display performed as an engineering evaluation by The NASA Johnson Space Center (JSC)-specifically. EMI. Thermal Vac, and radiation tests. The AMOLED display is an active-matrix Organic Light Emitting Diode (OLED) technology. The testing provided an initial understanding of the technology and its suitability for space applications. Relative to light emitting diode (LED) displays or liquid crystal displays (LCDs), AMOLED displays provide a superior viewing experience even though they are much lighter and smaller, produce higher contrast ratio and richer colors, and require less power to operate than LCDs. However, AMOLED technology has not been demonstrated in a space environment. Therefore, some risks with the technology must be addressed before they can be seriously considered for human spaceflight. The environmental tests provided preliminary performance data on the ability of the display technology to handle some of the simulated induced space/spacecraft environments that an AMOLED display will see during a spacecraft certification test program. This engineering evaluation is part of a Space Act Agreement (SM) between The NASA/JSC and Honeywell International (HI) as a collaborative effort to evaluate the potential use of AMOLED technology for future human spaceflight missions- both government-led and commercial. Under this SM, HI is responsible for doing optical performance evaluation, as well as temperature and touch screen studies. The NASA/JSC is responsible for performing environmental testing comprised of EMI, Thermal Vac, and radiation tests. Additionally, as part of the testing, limited optical data was acquired to assess performance as the display was subjected to the induced environments. The NASA will benefit from this engineering evaluation by understanding AMOLED suitability for future use in space as well as becoming a smarter buyer (or developer) of the technology. HI benefits

  20. Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation

    PubMed Central

    2014-01-01

    The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology. PMID:25341086

  1. 76 FR 44086 - Agency Information Collection (Report of Medical Examination for Disability Evaluation) Activity...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-22

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Agency Information Collection (Report of Medical Examination for Disability Evaluation) Activity.... 2900-0052.'' SUPPLEMENTARY INFORMATION: Title: Report of Medical Examination for Disability...

  2. 78 FR 26625 - Agency Information Collection Activities; Comment Request; Guaranty Agency Financial Report

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-07

    ... Management Services, Office of Management. BILLING CODE 4000-01-P ... Agency Information Collection Activities; Comment Request; Guaranty Agency Financial Report AGENCY... notice will be considered public records. Title of Collection: Guaranty Agency Financial Report....

  3. Novel reporter gene expression systems for monitoring activation of the Aspergillus nidulans HOG pathway.

    PubMed

    Furukawa, Kentaro; Yoshimi, Akira; Furukawa, Takako; Hoshi, Yukiko; Hagiwara, Daisuke; Sato, Natsuko; Fujioka, Tomonori; Mizutani, Osamu; Mizuno, Takeshi; Kobayashi, Tetsuo; Abe, Keietsu

    2007-07-01

    The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a beta-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogADelta. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogADelta mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds. PMID:17617716

  4. Final Progress Report for the activity called AMO2010 committee

    SciTech Connect

    Donald Shapero; Michael Moloney

    2006-12-31

    The committee was charged to produce a comprehensive report on the status of AMO Science. The committee was charged to produce a report that: 1. Reviewed the field of AMO science, emphasize recent accomplishments, and identify new opportunities and compelling scientific questions; 2. Identified the impact of AMO science on other scientific fields, emerging technologies, and national needs; 3. Identified future workforce, societal and educational needs for AMO science; and 4. Made recommendations on how the US research enterprise might realize the full potential of AMO science. The committee also produced an intermediate report addressing key research issues and themes facing the research community.

  5. Activity Sourcebook for Earth Science. Science Education Information Report.

    ERIC Educational Resources Information Center

    Mayer, Victor J., Ed.

    Designed to provide teachers of earth science with activities and information that will assist them in keeping their curricula up to date, this publication contains activities grouped into six chapters. Chapter titles are: (1) Weather and Climate, (2) Oceans, (3) The Earth and Its Surface, (4) Plate Tectonics, (5) Uses of Space Photography, and…

  6. Environmental Activities for Teaching Critical Thinking. [Environmental Education Information Report.

    ERIC Educational Resources Information Center

    Howe, Robert W.; Disinger, John F.

    The ability to think critically is essential if individuals are to live, work, and function effectively in our current and changing society. The activities included in this publication were selected to identify a variety of effective strategies for teaching critical thinking skills through environmental education. Activities include library…

  7. Albeni Falls Wildlife Mitigation Project : Annual Report of Mitigation Activities.

    SciTech Connect

    Entz, Ray D.

    2001-04-01

    The Albeni Falls Interagency Work Group was actively involved in implementing wildlife mitigation activities in 2000. The Work Group met each quarter to discuss management and budget issues affecting Albeni Falls wildlife mitigation. Members of the Work Group protected a total of 1,242 acres of wetland habitat in 2000. The total amount of wildlife habitat protected for Albeni Falls mitigation is approximately 4,190 acres (4,630 Habitat Units). Approximately 16% of the total wildlife habitat lost has been mitigated. Land management activities were limited in 2000 as protection opportunities took up most staff time. Administrative activities increased in 2000 as funding was more evenly distributed among Work Group members. As a result, implementation is expected to continue to increase in the coming year. Land management and monitoring and evaluation activities will increase in 2001 as site-specific management plans are completed and implemented.

  8. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS. PMID:25500996

  9. 30 CFR 251.8 - Inspection and reporting requirements for activities under a permit.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... activities. You must allow MMS representatives to inspect your exploration or scientific research activities... final report of exploration or scientific research activities under a permit within 30 days after the... and blocks in which any exploration or permitted scientific research activities were...

  10. Regulation of Activator Protein-1 by 8-iso-Prostaglandin E{sub 2} in a Thromboxane A{sub 2} Receptor-Dependent and -Independent Manner

    SciTech Connect

    Weber, Thomas J.; Markillie, Lye MENG.

    2003-05-01

    The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP isoforms may be regulated by novel ligands termed the isoprostanes, which paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes (8-iso-PG{sub 2{sub {alpha}}} and 8-iso-PGE{sub 2}) regulate the activity of TP isoforms expressed in Chinese Hamster Ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist (U46619) in CHO cells transfected with the human TP-P and TP-E receptors and this response was fully inhibited by TP antagonists (ISAP, SQ29,548). AP-1-luciferase activity was potently (nM) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, while 8-iso-PGF{sub 2{sub {alpha}}} was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1-10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.

  11. Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

    NASA Astrophysics Data System (ADS)

    Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

    2012-06-01

    Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

  12. Activities Underway To Improve Teacher Training, but Reporting on These Activities Could Be Enhanced. Report to Congressional Committees.

    ERIC Educational Resources Information Center

    Ashby, Cornelia M.

    This report examines two components of federal legislation to enhance teaching quality by improving the training of prospective teachers and the qualifications of current teachers. One component provides grants, and the other (the accountability provisions) requires collecting and reporting information the quality of teacher training programs and…

  13. Design for efficient Suburban Activity Centers. Phase 1 report

    SciTech Connect

    1996-11-19

    The advent of Suburban Activity Centers has had a radical effect on the shape and function of regions throughout the country. These centers are typically made up of large concentrations of office space, retail uses, and more recently, light industrial and manufacturing facilities. Very few Suburban Activity Centers include significant residential components, much less parks, schools, and other civic buildings. While SACs come in many sizes and shapes, there appear to be a number of distinctive common characteristics. The overall purpose of the study is to identify methods for designing Activity Centers so that they minimize traffic congestion, improve pedestrian, bicycle, and transit model shares and contribute to healthy regions.

  14. Activities report of the Institute of Sound and Vibration Research

    NASA Astrophysics Data System (ADS)

    Research concerning fluid dynamics and acoustics; audiology and human effects; structures and machinery; and signal processing and control is summarized. Aircraft noise; underwater acoustics; silencers; biomechanics; noise measurement; hearing; structural dynamics; laser technology; automotive engineering; and active control are discussed.

  15. Rural Enterprises, Incorporated report of significant activities and accomplishments

    NASA Technical Reports Server (NTRS)

    1985-01-01

    The ongoing activities of Rural Enterprises, Inc. are presented. The function of Rural Enterprises is to bring innovation from its rudimentary conceptual stages to useful and productive ends by means of cooperation with government, business, and educational institutions.

  16. National Alliance of Clean Energy Incubator Activities - Final Technical Report

    SciTech Connect

    Chris Downing, P.E.

    2004-12-14

    Summary of activity related to development of the Alliance of Clean Energy Business Incubators and incubation services provided to the clean energy sector by the Advanced Technology Development Center at the Georgia Institute of Technology.

  17. Report on Active and Planned Spacecraft and Experiments

    NASA Technical Reports Server (NTRS)

    Vostreys, R. W. (Editor); Maitson, H. H. (Editor)

    1981-01-01

    Active and planned spacecraft activity and experiments between June 1, 1980 and May 31, 1981 known to the National Space Science Data Center are described. The information covers a wide range of disciplines: astronomy, Earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. Each spacecraft and experiment is described and its current status presented. Descriptions of navigational and communications satellites and of spacecraft that contain only continuous radio beacons used for ionospheric studies are specifically excluded.

  18. A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome

    PubMed Central

    Coelho, Adriano C.; Oliveira, Jordana C.; Espada, Caroline R.; Reimão, Juliana Q.; Trinconi, Cristiana T.; Uliana, Silvia R. B.

    2016-01-01

    Background Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine. Methodology/Principal Findings A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine. Conclusions/Significance Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration. PMID:27144739

  19. Assessing adult leisure activities: an extension of a self-report activity questionnaire.

    PubMed

    Jopp, Daniela S; Hertzog, Christopher

    2010-03-01

    Everyday leisure activities in adulthood and old age have been investigated with respect to constructs such as successful aging, an engaged lifestyle, and prevention of age-related cognitive decline. They also relate to mental health and have clinical value, as they can inform diagnosis and interventions. In the present study, the authors enhanced the content validity of the Victoria Longitudinal Study activity questionnaire by adding items on physical and social activities and validated a shortened version of the questionnaire. The proposed leisure activity model included 11 activity categories: 3 types of social activities (i.e., activities with close social partners, group-centered public activity, religious activities), physical activities, developmental activities, experiential activities, crafts, game playing, TV watching, travel, and technology use. Confirmatory factor analyses validated the proposed factor structure in 2 independent samples. A higher order model with a general activity factor fitted the activity factor correlations with relatively little loss of fit. Convergent and discriminant validity for the activity scales were supported by patterns of their correlations with education, health, depression, cognition, and personality. In sum, the scores derived from of the augmented Victoria Longitudinal Study activity questionnaire demonstrate good reliability, and validity evidence supports their use as measures of leisure activities in young, middle-aged, and older individuals. PMID:20230157

  20. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    PubMed

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene. PMID:26585143

  1. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  2. High energy physics division semiannual report of research activities

    SciTech Connect

    Schoessow, P.; Moonier, P.; Talaga, R.; Wagner, R. )

    1991-08-01

    This report describes the research conducted in the High Energy Physics Division of Argonne National Laboratory during the period of January 1, 1991--June 30, 1991. Topics covered here include experimental and theoretical particle physics, advanced accelerator physics, detector development, and experimental facilities research. Lists of division publications and colloquia are included.

  3. Active Ageing and Universities: Engaging Older Learners. Research Report

    ERIC Educational Resources Information Center

    Phillipson, Chris; Ogg, Jim

    2010-01-01

    This report reviews the engagement of older learners (defined as those aged 50 and over) in education and training with particular reference to their involvement in higher education. The ageing of populations was one of the most important trends in the 20th century and will raise major challenges in this century. Appended are: (1) Selected UK…

  4. BNL National Synchrotron Light Source activity report 1997

    SciTech Connect

    1998-05-01

    During FY 1997 Brookhaven National Laboratory celebrated its 50th Anniversary and 50 years of outstanding achievement under the management of Associated Universities, Inc. This progress report is divided into the following sections: (1) introduction; (2) science highlights; (3) meetings and workshops; (4) operations; (5) projects; (6) organization; and (7) abstracts and publications.

  5. Instructional Simulation: A Research Development and Dissemination Activity. Final Report.

    ERIC Educational Resources Information Center

    Twelker, Paul A., Ed.; And Others

    This report describes design techniques, areas of effective application, and research directions in educational simulations. The five chapters contain (1) a review of recent literature; (2) an overview of the field of simulation including definitions and some of the rationales for using simulation in instruction; (3) an outline of the design…

  6. 12 CFR 21.11 - Suspicious Activity Report.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...CEN means the Financial Crimes Enforcement Network of the Department of the Treasury. (2) Institution... the suspect or group of suspects, as well as alias identifiers, such as drivers' license or social... pursuant to the reporting requirements of 17 CFR 240.17f-1. (g) Retention of records. A national bank...

  7. 12 CFR 21.11 - Suspicious Activity Report.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. A SAR, and any... pursuant to the reporting requirements of 17 CFR 240.17f-1. (g) Retention of records. A national bank shall... CFR 4.33. (l) Limitation on liability. A national bank and any director, officer, employee or agent...

  8. 12 CFR 21.11 - Suspicious Activity Report.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. A SAR, and any... pursuant to the reporting requirements of 17 CFR 240.17f-1. (g) Retention of records. A national bank shall... CFR 4.33. (l) Limitation on liability. A national bank and any director, officer, employee or agent...

  9. 12 CFR 21.11 - Suspicious Activity Report.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... pursuant to the reporting requirements of 17 CFR 240.17f-1. (g) Retention of records. A national bank shall... appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. A SAR, and any... CFR 4.33. (l) Limitation on liability. A national bank and any director, officer, employee or agent...

  10. 12 CFR 21.11 - Suspicious Activity Report.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... pursuant to the reporting requirements of 17 CFR 240.17f-1. (g) Retention of records. A national bank shall... appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. SARs are confidential... disclosure of non-public OCC information under 12 CFR 4.33. (l) Limitation on liability. A national bank...

  11. Annual Report on LSCA Special Activities, FY 1983.

    ERIC Educational Resources Information Center

    Neff, Evaline; And Others

    This compilation results from efforts of the State and Public Library Services Branch of the U.S. Department of Education to disseminate pertinent information submitted by the State Library Administrative Agencies on key LSCA (Library Services and Construction Act) program areas. Each report is written by an administrative librarian responsible…

  12. DOE and AID stand-alone photovoltaic activities. Status report

    SciTech Connect

    Bifano, W.J.; Ratacajczak, A.F.

    1983-01-01

    The NASA Lewis Research Center (LeRC) is managing stand-alone photovoltaic (PV) system activities sponsored by the US Department of Energy (DOE) and the US Agency for International Development (AID). The DOE project includes village PV power demonstration projects in Gabon (four sites) and the Marshall Islands, PV-powered medical refrigerators in six countries, PV system microprocessor control development activities and PV-hybrid system assessments. The AID project includes a large village systen in Tunisia, a water pumping/grain grinding project in Upper Volta, five medical clinics in four countries, PV-powered medical refrigerator field tests in eighteen countries and one PV-powered remote earth station application. This paper reviews these PV activities and summarizes significant findings to date.

  13. Report on Second Activations with the Lead Slowing Down Spectrometer

    SciTech Connect

    Stave, Sean C.; Mace, Emily K.; Pratt, Sharon L.; Warren, Glen A.

    2012-04-27

    Summary On August 18 and 19 2011, five items were irradiated with neutrons using the Lead Slowing Down Spectrometer (LSDS). After irradiation, dose measurements and gamma-spectrometry measurements were completed on all of the samples. No contamination was found on the samples, and all but one provided no dose. Gamma-spectroscopy measurements qualitatively agreed with expectations based on the materials. As during the first activation run, we observed activation in the room in general, mostly due to 56Mn and 24Na. Most of the activation of the samples was short lived, with half-lives on the scale of hours to days, except for 60Co which has a half-life of 5.3 y.

  14. Teaching Activity Report: A Concise and Simple Summary of Individual Instructional Effort. AIR Forum 1979 Paper.

    ERIC Educational Resources Information Center

    McCollester, Charles W.; Farrell, Richard L., Jr.

    The University of Notre Dame developed the Teaching Activity Report in an effort to present professors with meaningful summaries of their individual instructional efforts each semester. Geared to meet the needs of the individual instructors and their superiors, the Teaching Activity Report synthesizes all facets of the individual's instructional…

  15. 33 CFR 6.16-1 - Reporting of sabotage and subversive activity.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Reporting of sabotage and... HOMELAND SECURITY GENERAL PROTECTION AND SECURITY OF VESSELS, HARBORS, AND WATERFRONT FACILITIES Sabotage and Subversive Activity § 6.16-1 Reporting of sabotage and subversive activity. Evidence of...

  16. Reliability and Validity of a New Physical Activity Self-Report Measure for Younger Children

    ERIC Educational Resources Information Center

    Belton, Sarahjane; Mac Donncha, Ciaran

    2010-01-01

    The purpose of this study was to assess the test-retest reliability and validity of a new Youth Physical Activity Self-Report measure. Heart rate and direct observation were employed as criterion measures with a sample of 79 children (aged 7-9 years). Spearman's rho correlation between self reported activity intensity and heart rate was 0.87 for…

  17. Apollo experience report: Assessment of metabolic expenditures. [extravehicular activity

    NASA Technical Reports Server (NTRS)

    Waligora, J. M.; Hawkins, W. R.; Humbert, G. F.; Nelson, L. J.; Vogel, S. J.; Kuznetz, L. H.

    1975-01-01

    A significant effort was made to assess the metabolic expenditure for extravehicular activity on the lunar surface. After evaluation of the real-time data available to the flight controller during extravehicular activity, three independent methods of metabolic assessment were chosen based on the relationship between heart rate and metabolic production, between oxygen consumption and metabolic production, and between the thermodynamics of the liquid-cooled garment and metabolic production. The metabolic assessment procedure is analyzed and discussed. Real-time use of this information by the Apollo flight surgeon is discussed. Results and analyses of the Apollo missions and comments concerning future applications are included.

  18. Report on First Activations with the Lead Slowing Down Spectrometer

    SciTech Connect

    Warren, Glen A.; Mace, Emily K.; Pratt, Sharon L.; Stave, Sean; Woodring, Mitchell L.

    2011-03-03

    On Feb. 17 and 18 2011, six items were irradiated with neutrons using the Lead Slowing Down Spectrometer. After irradiation, dose measurements and gamma-spectrometry measurements were completed on all of the samples. No contamination was found on the samples, and all but one provided no dose. Gamma-spectroscopy measurements qualitatively agreed with expectations based on the materials, with the exception of silver. We observed activation in the room in general, mostly due to 56Mn and 24Na. Most of the activation was short lived, with half-lives on the scale of hours, except for 198Au which has a half-life of 2.7 d.

  19. Aeronautics and space report of the president, 1974 activities

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The U.S. Government activities for 1974 in aeronautics and space are presented. Significant contributions toward the fulfillment of the nation's goals in space and aeronautics are covered, including application of space systems and technology to beneficial uses on earth, exploration of space and increase of scientific knowledge, development of improved space systems and technology, international cooperation, and advancement of civil and military aeronautics. Also in 1974, space activities in the private sector expanded to provide additional services to the public. The accomplishments are summarized.

  20. Assessing Adult Leisure Activities: An Extension of a Self-Report Activity Questionnaire

    PubMed Central

    Jopp, Daniela; Hertzog, Christopher

    2009-01-01

    Everyday leisure activities in adulthood and old age have been investigated with respect to constructs such as successful aging, an engaged lifestyle, and prevention of age-related cognitive decline. They also relate to mental health and have clinical value as they can inform diagnosis and interventions. In the present study, we enhanced the content validity of the Victoria Longitudinal Study activity questionnaire by adding items on physical and social activities, and validated a shortened version of the questionnaire. Our proposed leisure activity model included 11 activity categories: three types of social activities (i.e., activities with close social partners, group-centered public activity, religious activities), physical, developmental, and experiential activities, crafts, game playing, TV watching, travel, and technology use. Confirmatory factor analyses validated the proposed factor structure in two independent samples. A higher-order model with a general activity factor fitted the activity factor correlations with relatively little loss of fit. Convergent and discriminant validity for the activity scales were supported by patterns of their correlations with education, health, depression, cognition, and personality. In sum, the scores derived from of the augmented VLS activity questionnaire demonstrate good reliability, and validity evidence supports their use as measure of leisure activities in young, middle-aged, and older individuals. PMID:20230157

  1. Report on active and planned spacecraft and experiments

    NASA Technical Reports Server (NTRS)

    Littlefield, R. G. (Editor)

    1983-01-01

    Information concerning active and planned spacecraft and experiments is included. The information covers a wide range of scientific disciplines: astronomy, earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. These spacecraft projects represent the efforts and fundng of individual countries as well as cooperative arrangements among different countries.

  2. Activities report of the Institute of Sound and Vibration Research

    NASA Astrophysics Data System (ADS)

    Research in fluid dynamics and acoustics (noise and vibration control); audiology and human effects (audiotory communication and hearing conservation); structures and machinery (automotive design); and shock analysis is summarized. Underwater acoustics; active noise control; aircraft noise; wind turbine noise; laminar flow fans; helmet design; and the acoustics of flow ducts were studied.

  3. Gross Activity of Children at Play. (Internal Report).

    ERIC Educational Resources Information Center

    Wuellner, Lance

    Time-lapse photography was used to record the gross play activity of preschool children, rated according to three measures of equipment use and three measures of movement. The definition and derivation of these measures was outlined, and five hypotheses were presented and tested concerning the variability and interrelation of the measures.…

  4. 11 CFR 300.36 - Reporting Federal election activity; recordkeeping.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... political committee (see 11 CFR 100.5). Such a party committee or association of candidates or officeholders... political committee under 11 CFR 100.5, unless the payment otherwise qualifies as an expenditure under 2 U.S... funds or Levin funds for Federal election activity (see 11 CFR 300.32 and 300.33) by either such a...

  5. Active system area networks for data intensive computations. Final report

    SciTech Connect

    2002-04-01

    The goal of the Active System Area Networks (ASAN) project is to develop hardware and software technologies for the implementation of active system area networks (ASANs). The use of the term ''active'' refers to the ability of the network interfaces to perform application-specific as well as system level computations in addition to their traditional role of data transfer. This project adopts the view that the network infrastructure should be an active computational entity capable of supporting certain classes of computations that would otherwise be performed on the host CPUs. The result is a unique network-wide programming model where computations are dynamically placed within the host CPUs or the NIs depending upon the quality of service demands and network/CPU resource availability. The projects seeks to demonstrate that such an approach is a better match for data intensive network-based applications and that the advent of low-cost powerful embedded processors and configurable hardware makes such an approach economically viable and desirable.

  6. 12 CFR 208.62 - Suspicious activity reports.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... transaction related to a money laundering activity or a violation of the Bank Secrecy Act. This section... or more that involve potential money laundering or violations of the Bank Secrecy Act. Any... 17 CFR 240.17f-1. (g) Retention of records. A member bank shall maintain a copy of any SAR filed...

  7. Materials and Activities for Teachers and Children (MATCH). Program Report.

    ERIC Educational Resources Information Center

    Bye, Margaret

    Self contained multi-media kits for grades 1 through 6 involve students directly in the learning process. Emphasis is on non-verbal learning which takes place when youngsters examine real objects and engage in learning activities. Involved in the discovery and inquiry process, students hypothesize, classify, and categorize. In an interdisciplinary…

  8. Activities report of the Institute for Extraterrestrial Physics

    NASA Astrophysics Data System (ADS)

    1990-01-01

    The fields of activity are divided into four parts: in situ space measurements; astrophysical measurements; laboratory measurements; and theory. The Institute's denomination originates for the main subject of research, space research, and from the research method. Articles dealing with near Earth space and solar system, infrared and submillimeter astronomy, x ray astronomy, gamma astronomy and laboratory astrophysics are covered.

  9. Report on active and planned spacecraft and experiments

    NASA Technical Reports Server (NTRS)

    Horowitz, R. (Editor); Nostreys, R. W. (Editor)

    1980-01-01

    Information on current and planned spacecraft activity for a broad range of scientific disciplines is presented. The information covers a wide range of disciplines: astronomy, Earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. These spacecraft projects represent the efforts and funding of individual countries as well as cooperative arrangements among different countries.

  10. Report on active and planned spacecraft and experiments

    NASA Technical Reports Server (NTRS)

    Vette, J. I. (Editor); Vostreys, R. W. (Editor); Horowitz, R. (Editor)

    1978-01-01

    Information is presented, concerning active and planned spacecraft and experiments known to the National Space Science Data Center. The information included a wide range of disciplines: astronomy, earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. These spacecraft projects represented the efforts and funding of individual countries as well as cooperative arrangements among different countries.

  11. Report on active and planned spacecraft and experiments

    NASA Technical Reports Server (NTRS)

    Brecht, J. J. (Editor)

    1974-01-01

    Information dealing with active and planned spacecraft and experiments known to the National Space Science Data Center (NSSDC) is presented. Included is information concerning a wide range of disciplines: astronomy, earth sciences, meteorology, planetary sciences, aeronomy, particles and fields, solar physics, life sciences, and material sciences. These spacecraft represent the efforts and funding of individual countries, as well as cooperative arrangements among different countries.

  12. Caregiver Reports of Children's Activity Participation Following Serious Injury.

    PubMed

    Braaf, Sandra; Ameratunga, Shanthi; Teague, Warwick; Jowett, Helen; Gabbe, Belinda

    2016-01-01

    Paediatric trauma can result in significant levels of on-going disability. The aim of this study was to explore the restrictions on activity participation that children experience following serious injury from the perspective of their caregivers. We performed a thematic analysis of transcripts of semi-structured in-depth interviews with the caregivers of 44 seriously injured children, conducted three-years after the injury, and purposively sampled from a population-based cohort study. Both temporary and on-going restrictions on school, sport, leisure and social activities were identified, some of which were imposed by caregivers, schools, or recommended by health providers. The perceived risk of further injury, physical restrictions, emotional state and fatigue levels were important influences on degrees of activity restriction. Children who were socially less engaged, especially those who were more severely injured, had difficulty making and retaining friends, and exhibited signs of depression or social withdrawal. The activities of pre-school children were strongly regulated by their caregivers, while school age children faced obstacles with participation in aspects such as study, sport, and peer and teacher relationships, affecting learning, school attendance and enjoyment. The findings highlight the need for primary prevention and reducing the impacts of serious injury throughout the continuum of care. PMID:27399741

  13. Activation of the rainbow trout metallothionein-A promoter by silver and zinc.

    PubMed

    Mayer, Gregory D; Leach, Allan; Kling, Peter; Olsson, Per-Erik; Hogstrand, Christer

    2003-01-01

    In fish, the synthesis of metallothionein (MT) is increased by a number of heavy metals. The rainbow trout MT-A gene promoter region contains six known metal responsive elements (MREs), that mediate promoter activation by metals. In the present study, two fish cell lines differing in their ability to produce MT, RTG-2 (produce MT protein) and CHSE-214 (produce no detectable MT protein), were used to help elucidate the roles of Zn, Ag and MT in the activation of the MT promoter. The hypothesis tested was that Ag activates the MT-A promoter indirectly by displacing Zn from pre-existing Zn-MT and that this liberated Zn subsequently induces MT synthesis. Both cell lines were transfected with a luciferase reporter gene construct containing the rainbow trout MT-A promoter, exposed to various concentrations of Zn or Ag, and assayed for luciferase activity. CHSE-214 cells showed five times greater production of luciferase than RTG-2 cells when exposed to identical concentrations of Ag. Thus, Ag can likely induce MT transcription without displacing Zn from pre-existing Zn-MT. Furthermore, Ag activated the MT promoter at concentrations 100-fold lower than those required for Zn to initiate transcription, suggesting that zinc displaced from other sites by such low concentrations of Ag would not be sufficient to initiate MT transcription. This interpretation was further supported by radiotracer studies indicating that Ag did not cause a redistribution of 65Zn within either of the two cell types. These combined results indicate that Ag may be a direct inducer of MT. PMID:12524046

  14. HAL/S-360 compiler test activity report

    NASA Technical Reports Server (NTRS)

    Helmers, C. T.

    1974-01-01

    The levels of testing employed in verifying the HAL/S-360 compiler were as follows: (1) typical applications program case testing; (2) functional testing of the compiler system and its generated code; and (3) machine oriented testing of compiler implementation on operational computers. Details of the initial test plan and subsequent adaptation are reported, along with complete test results for each phase which examined the production of object codes for every possible source statement.

  15. DDE-MURR Status Report of Conceptual Design Activities

    SciTech Connect

    N.E. Woolstenhulme; R.B. Nielson; M.H. Sprenger; G.K. Housley

    2013-09-01

    The Design Demonstration Experiment for the University of Missouri Research Reactor (DDE-MURR) is intended to facilitate Low Enriched Uranium (LEU) conversion of the MURR by demonstrating the performance and fabrication of the LEU fuel element design through an irradiation test in a 200mm channel at the Belgium Reactor 2 (BR2). Revision 0 of this report was prepared at the end of government fiscal year 2012 when most of the resources for furthering DDE design work were expected to be postponed. Hence, the conceptual design efforts were summarized to provide the status of key objectives, notable results, and provisions for future design work. Revision 1 of this report was prepared at the end of fiscal year 2013 in order to include results from a neutronic study performed by BR2, to incorporate further details that had been achieved in the engineering sketches of the irradiation devices, and to provide an update of the DDE-MURR campaign in relation to program objectives and opportunities for its eventual irradiation. These updates were purposed to bring the DDE-MURR conceptual design to level of maturity similar to that of the other two DDE efforts (DDE-MITR and DDE-NBSR). This report demonstrates that the DDE-MURR design effort is well on the path to producing a suitable irradiation experiment, but also puts forth several recommendations in order to facilitate success of the irradiation campaign.

  16. TUNL XIX. Annual report, January 1-December 31, 1980. [North Carolina State Univ. activities at TUNL

    SciTech Connect

    1980-12-31

    Research performed by North Carolina State University personnel at TUNL is highlighted in this report, which is actually the complete TUNL progress report for 1980. Studies in the areas of neutron cross sections, neutron polarization, radiative capture, atomic physics and development activities are included. One may expect completed projects to be reported in physics journals or conference proceedings. (RWR)

  17. 76 FR 31684 - Agency Information Collection (Medical Expense Report) Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-01

    ... AFFAIRS Agency Information Collection (Medical Expense Report) Activity Under OMB Review AGENCY: Veterans...: Medical Expense Report, VA Form 21-8416. OMB Control Number: 2900-0161. Type of Review: Extension of a... income-based benefits to report medical expenses paid. Unreimbursed medical expenses may be excluded...

  18. The PPAR{gamma} ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

    SciTech Connect

    Moss, Patrice E.; Lyles, Besstina E.; Stewart, LaMonica V.

    2010-12-10

    The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPAR{gamma} ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPAR{gamma} ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPAR{gamma} ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPAR{gamma}. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPAR{gamma} and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.

  19. STS-99 Crew Activities Report/Flight Day 1 Highlights

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Live footage shows the crew, Commander Kevin R. Kregel, Pilot Dominic L. Pudwill Gorie, and Mission Specialists Janet L. Kavandi, Janice E. Voss, Mamoru Mohri and Gerhard P.J. Thiele, seated in the dining room with the traditional cake. The crew is seen performing various pre-launch activities including suit-up, walk out to the Astro-van, and strap-in into the vehicle. Also seen are the retractions of the orbiter access arm and the gaseous oxygen mint hood, main engine start, booster ignition, liftoff, and separation of the solid rocket boosters. The Red Team (first of the dual shift crew) includes Kregel, Kavandi, and Thiele, who are shown conducting jet thruster firings, activating radar instruments, and deploying the boom (mass).

  20. STS-106 Crew Activity Report/Flight Day 1 Highlights

    NASA Technical Reports Server (NTRS)

    2000-01-01

    On this first day of the STS-106 Atlantis mission, the flight crew, Commander Terrence W. Wilcutt, Pilot Scott D. Altman, and Mission Specialists Daniel C. Burbank, Edward T. Lu, Richard A. Mastracchio, Yuri Ivanovich Malenchenko, and Boris V. Morukov are seen performing pre-launch activities. They are shown sitting around the breakfast table with the traditional cake, suiting-up, and riding out to the launch pad. The final inspection team is seen as they conduct their final check of the space shuttle on the launch complex. Also, included are various panoramic views of the shuttle on the pad. The crew is readied in the 'white room' for their mission. After the closing of the hatch and arm retraction, launch activities are shown including countdown, engine ignition, launch, and the separation of the Solid Rocket Boosters.